Anti-cancer peptide JZTX-66 derived from chilobrachys guangxiensis venom and application of anti-cancer peptide JZTX-66
By isolating and identifying the anticancer peptide JZTX-66 from the venom of the spider *Amanita muscaria*, the problem of the lack of sustained efficacy and significant side effects in existing anticancer treatments has been solved, achieving highly efficient killing and inhibition of mouse melanoma cells.
Patent Information
- Application Number
- CN202511165302.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-20
- Publication Date
- 2025-11-18
AI Technical Summary
Existing clinical radiotherapy and chemotherapy have short-lasting effects and significant side effects in cancer treatment, necessitating the development of new, safe, and highly effective anticancer drugs.
An anticancer peptide, JZTX-66, was isolated and identified from the venom of the spider *Sargassum fusiforme*. The venom was collected by electrical stimulation, purified by RP-HPLC, and its amino acid sequence was determined by Edman degradation and in-solution enzymatic digestion-desalting-LC-MS/MS. The peptide sample was prepared by prokaryotic expression, and its broad-spectrum anticancer activity was verified by in vitro activity experiments.
JZTX-66 exhibits strong cytotoxic activity against mouse melanoma cells B16-F10, inducing apoptosis, disrupting cell membranes, and inhibiting proliferation and migration. Furthermore, metal cations such as Ca2+ can reverse its cytotoxic activity.
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Abstract
Description
Technical Field
[0001] This invention relates to the screening, preparation and application of anticancer peptides, belonging to the field of biomedicine, specifically to the preparation and application of an anticancer peptide JZTX-66 derived from the venom of the spider *Gnaphalium affineum*. Background Technology
[0002] Cancer remains a major public health problem worldwide, and current clinical radiotherapy and chemotherapy methods often fail to provide sustained anti-cancer effects, even producing serious side effects and drug resistance. Therefore, developing novel, safe, and effective anti-cancer drugs is of paramount importance. Natural toxins derived from venomous animals are a valuable resource for developing novel anti-cancer drugs. This invention isolates and identifies a novel anti-cancer peptide, JZTX-66, from the venom of the spider *Amanita muscaria*, which can be applied to the development of anti-tumor drugs and exhibits strong anti-cancer activity. Summary of the Invention
[0003] The purpose of this invention is to provide a bioactive peptide with anticancer activity isolated and identified from the venom of the spider *Sargassum fusiforme*, and to discover that the anticancer peptide JZTX-66 has broad-spectrum anticancer activity, and exhibits strong cytotoxic activity against mouse melanoma cells B16-F10. It also demonstrates multiple anticancer effects, including inducing apoptosis at high concentrations, directly disrupting the cell membrane leading to cell death, and inhibiting the proliferation, migration, and invasion of B16-F10 cells at low concentrations. 2+ Metal cations can inhibit the cytotoxic activity of JZTX-66, and this inhibition can be reversed by EDTA.
[0004] To achieve the above objectives, the technical solution adopted in this invention is as follows: using the *Jingzhao* spider as the research object, spider venom is obtained through electrical stimulation, and the target polypeptide JZTX-66 is separated and purified by RP-HPLC technology. The target polypeptide contains 110 amino acid residues and is composed of three parts: a signal peptide, a precursor peptide, and a mature peptide. The mature peptide region consists of 66 amino acid residues.
[0005] Further verification of the molecular weight and primary structure of the mature peptide was performed using Edman degradation and in-solution enzymatic digestion-desalting-LC-MS / MS techniques. The molecular weight of the target peptide was determined to be 6766.1643 Da, with an amidation modification at the C-terminus. Its amino acid sequence is as follows: GDDGTCILKGDHCHGTCDCCGWTTTCRKSSKSAGGKICKSEGSSISAFNAIAKGVAAMKKAKCKHKS.
[0006] Further in vitro activity experiments revealed that it possesses broad-spectrum anticancer activity and exhibits strong cytotoxic activity against mouse melanoma cells B16-F10, inducing cell death in various ways. Therefore, the anticancer peptide JZTX-66 derived from the venom of the spider *Gnaphalium affine* can be used as a potential anticancer drug. Attached Figure Description
[0007] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0008] Figure 1 Separation, purification, and activity identification of JZTX-66. A, RP-HPLC chromatogram (215 nm) of crude venom from *Amanita ventricosa* (Jingzhao hairy spider), with the inset showing the morphology of *Amanita ventricosa*. The positions indicated by the red asterisks are the crude components of the target molecule. B, Second-round fine RP-HPLC chromatogram (215 nm) of the components marked by red asterisks in Figure A, with the red asterisks representing single target molecule components. C, Cytotoxicity analysis of the natural target components after fine separation against B16-F10.
[0009] Figure 2 Images of JZTX-66 prokaryotic expression and anticancer activity identification. A, SDS-PAGE of JZTX-66 prokaryotic expression, M is the protein marker, lane 1 is the pre-induction bacterial culture, and lane 2 is the post-induction bacterial culture. B, Chromatogram of prokaryotic expression after enzyme digestion (embedded fine separation map). C, Mass spectrometry identification of prokaryotic expression. D, Chromatograms of natural JZTX-66, prokaryotically expressed JZTX-66 (rJZTX-66), and a mixture of the two. E, Viability of B16-F10 cells treated with rJZTX-66.
[0010] Figure 3 JZTX-66 activity against different cancer cells and non-cancer cells.
[0011] Figure 4Cytotoxic activity analysis of JZTX-66 against B16-F10 cells under different culture medium conditions. A, Activity analysis of JZTX-66 against B16-F10 cells in 1640 and 1640+G (G is added glucose) media. BC, Tolerance analysis of B16-F10 cells at 12 h and 24 h in different specific culture media (a, DMEM medium; b, 1640 medium; c, DMEM medium containing 0.02% EDTA; d, DMEM medium containing 0.04% EDTA; e, DMEM medium containing 0.08% EDTA). D, Activity analysis of JZTX-66 against B16-F10 cells under different specific culture medium conditions (12 h).
[0012] Figure 5 Ca 2+ The effect of different culture media on the cytotoxic activity of JZTX-66 cells is shown in the figure. A and B represent the tolerance of B16-F10 cells to different specific culture media at 12 h and 24 h (a, DMEM medium; b, calcium-free DMEM medium; c, DMEM medium containing 0.2 mM Ca). 2+ Calcium-free DMEM medium; d, containing 0.4 mM Ca 2+ Calcium-free DMEM medium; e, containing 0.8 mM Ca 2+ Calcium-free DMEM medium; f, containing 1.8 mM Ca 2+ Calcium-free DMEM medium; g, containing 2 mM Ca 2+ (Calcium-free DMEM medium). C, JZTX-66 in different concentrations of Ca. 2+ Cytotoxic activity analysis in calcium-free DMEM medium (12 h). DE, analysis of tolerance of B16-F10 cells to different specific media at 12 h and 24 h (a, 1640 medium; b, medium containing 0.8 mM Ca). 2+ 1640 medium; c, containing 1.8 mM Ca 2+ 1640 culture medium; d, containing 2 mM Ca 2+ 1640 medium; e, containing 4 mM Ca 2+ 1640 medium). F, JZTX-66 in different concentrations of Ca. 2+ Cytotoxic activity analysis in 1640 medium (12 h).
[0013] Figure 6Detection diagram of apoptosis in B16-F10 cells using JZTX-66.
[0014] Figure 7 Figure A shows the effect of JZTX-66 on the proliferation of B16-F10 cells. A) Morphological characterization of B16-F10 cell colonies treated with different concentrations of JZTX-66. B) Statistical graph showing the effect of different concentrations of JZTX-66 on the formation of B16-F10 cell colonies.
[0015] Figure 8 The graph shows the detection of the cell cycle in B16-F10 cells using JZTX-66. A, Histogram of DNA content distribution. B, Bar chart of cell cycle distribution.
[0016] Figure 9 Figure 1 shows the effects of JZTX-66 on the migration and invasion of B16-F10 cells. A) Migration morphology of B16-F10 cells after treatment with different concentrations of JZTX-66. B) Invasion morphology of B16-F10 cells after treatment with different concentrations of JZTX-66. Statistical graph showing the effects of JZTX-66 on the migration (C) and invasion (D) of the B16-F10 cell line.
[0017] Figure 10 Characterization of cell membrane disruption by JZTX-66. SEM images (A), trypan blue staining (B), and SYTO9 / PI double staining (C) of B16-F10 cells treated with 5 μM and 10 μM JZTX-66 for 1 h in 1640 medium.
[0018] Figure 11 Analysis of the therapeutic effect of EDTA-assisted JZTX-66 on tumors in mice. A, Flowchart of JZTX-66 treatment of tumor-bearing mice. B, Tumors in mice were continuously treated with the drug for 3 days, and tumor changes were observed for 11 days after treatment. Morphological characteristics of tumors in each group after dissection at the end of the treatment period. C, Changes in tumor volume in mice. D, Changes in body weight in mice. Detailed Implementation
[0019] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.
[0020] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.
[0021] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.
[0022] A polypeptide of the present invention, which is derived from the active ingredient of the venom of the spider *Amanita muscaria*.
[0023] Research Results 1. Isolation, purification, and activity analysis of anticancer peptides derived from spider venom.
[0024] Using the melanoma cell line B16-F10 as the target, we screened the cytotoxic activity of nearly 10 kinds of crude spider venoms stored in our laboratory. Preliminary results showed that the crude venom of *Sphaeromorpha japonica* (also known as *Sphaeromorpha jingzhao*) exhibited the strongest activity. Therefore, further investigation is needed to explore the components responsible for its cytotoxic activity. The morphology of *Sphaeromorpha japonica* is as follows: Figure 1 As shown in Figure A. A certain amount of freeze-dried powder of the crude venom of *Sargassum fusiforme* was weighed and fully dissolved in water Q. The solution was filtered through a 0.22 μm filter to remove insoluble impurities such as dirt. Then, it was separated and purified using RP-HPLC technology. The RP-HPLC chromatogram of the crude venom is shown in Figure A. Figure 1 As shown in Figure A. Further cytotoxicity assays were performed on the collected chromatographic peaks, and the position marked with a red asterisk in the chromatogram was preliminarily identified as the target component. Sufficient amounts of the target component were collected and subjected to a second round of fine separation using a Waters chromatograph, as shown in Figure A. Figure 1 As shown in Figure B, the single chromatographic peak marked with a red asterisk is the target component. The target peak was collected, lyophilized, and weighed. Its IC50 value for action on B16-F10 cells was determined. 50 The value is 2.06 μM ( Figure 1 C).
[0025] 2. Prokaryotic expression and anticancer activity identification of JZTX-66.
[0026] JZTX-66 contains 66 amino acid residues. Considering its long peptide chain, the difficulty in obtaining samples through chemical synthesis, the large number of byproducts, and the fact that its four disulfide bonds are not conducive to its renaturation, we chose the *E. coli* prokaryotic expression system to prepare peptide samples. The target sequence of JZTX-66 was constructed into the pET 32a prokaryotic vector for prokaryotic expression, yielding a fusion protein containing JZTX-66. The fusion protein was then digested with TEV enzyme in a 16 °C water bath for 12–16 h. The fusion protein obtained from prokaryotic expression is shown below. Figure 2 The red dashed line in Figure A represents the target peak. After two rounds of RP-HPLC separation and purification, we obtained a relatively pure recombinant JZTX-66 (rJZTX-66) sample. The red asterisk in the figure marks the target peak. Figure 2 B). Simultaneously, the expressed pure sample was identified by MALDI-TOF mass spectrometry. Figure 2The results showed that its molecular weight of 6766.1431 Da was consistent with that of the naturally obtained JZTX-66 sample (6766.1643 Da). Furthermore, we performed chromatographic analysis on the natural JZTX-66 sample, the prokaryotically expressed rJZTX-66 sample, and a mixture of the two. The results showed that under the same elution gradient conditions, their peak shapes were basically identical, and their retention times were almost the same. Figure 2 D), indicating that the expressed polypeptide samples have the same structure and other properties. The IC50 of rJZTX-66 on B16-F10 cells... 50 The IC50 of natural JZTX-66 is 2.4 μM, while that of natural JZTX-66 is 2.06 μM. Figure 2 E), indicating that the activity of the JZTX-66 sample prepared by prokaryotic expression is consistent with that of the natural sample. For the convenience of peptide naming, all subsequent functional experiments used samples prepared by prokaryotic expression, and all were named JZTX-66.
[0027] 3. Cytotoxic activity analysis of JZTX-66 against multiple cell lines.
[0028] The above cytotoxic activity analysis showed that JZTX-66 has strong cytotoxic activity against the B16-F10 cell line. To test the broad spectrum of the peptide's effects, we further tested the cytotoxicity of JZTX-66 against 10 selected cancer cell lines and 5 non-cancer cell lines. The results are as follows. Figure 3 As shown, JZTX-66 exhibits weak activity against four non-cancer cell lines, suggesting a degree of selectivity. Regarding cancer cell lines, JZTX-66 shows weak activity against HeLa, A549, and CT26 cells, while its IC50 against the other seven selected cancer cell lines ranges from 2 to 6 μM, indicating a possible preference for cancer cell lines. Current results show that JZTX-66 exhibits the strongest cytotoxic activity against B16-F10 cells; therefore, subsequent functional experiments will be conducted using this cell line as a model.
[0029] 4. Effects of culture medium composition on the cytotoxic activity of JZTX-66 cells.
[0030] By comparing the differences in the main components of 1640 and DMEM media, and considering the trend of JZTX-66 cytotoxic activity against B16-F10 cells showing 1640 > DMEM, we identified the factors influencing activity as cations and glucose in the media. Firstly, the glucose content in the two media showed an opposite trend to the activity intensity. Therefore, we added additional glucose to the 1640 medium to achieve the same final concentration as in DMEM. Figure 4As shown in Figure A, the cytotoxic activity of JZTX-66 against B16-F10 was almost identical to that under 1640 medium conditions, ruling out the possibility that glucose affected the peptide. Secondly, the study showed that cations can competitively inhibit the binding of cationic antimicrobial peptides to bacteria, thereby affecting the antimicrobial activity of the peptides. Therefore, we utilized EDTA to chelate Ca in DMEM... 2+ Metal cations, to investigate Ca 2+ The effect of metal cations on peptide activity. For example... Figure 4 B and Figure 4 As shown in Figure C, we determined the optimal EDTA concentrations for cell treatment to be 0.02% and 0.04%, with a treatment time of 12 h. The cytotoxic activity of JZTX-66 against B16-F10 cells was tested by adding 0.02% and 0.04% EDTA to DMEM medium under these conditions. The results showed that the cytotoxicity of the peptide in DMEM with the addition of 0.04% EDTA was essentially the same as that in 1640 medium. Figure 4 D), the activity of JZTX-66 is affected by the Ca in the culture medium. 2+ The influence of metal cations, etc.
[0031] 5. Ca 2+ Effects on the cytotoxic activity of JZTX-66.
[0032] Ca 2+ The concentrations in both culture media were 0.4 mM (1640) < 1.8 mM (DMEM), which is the opposite trend in the cytotoxic activity of JZTX-66 under these conditions. To investigate Ca... 2+ To investigate the effect of peptide activity, we tested the cytotoxic activity of the peptide against B16-F10 cells under seven different culture conditions: DMEM medium, calcium-free DMEM medium, and DMEM medium supplemented with 0.2 mM, 0.4 mM, 0.8 mM, 1.8 mM, and 2 mM CaCl2, respectively. Incubation of B16-F10 cells in these media for 12 h showed good cell condition and unaffected viability, while incubation for 24 h significantly inhibited cell viability. Figure 5 A and Figure 5 (B), therefore, the incubation time between the drug and cells for cytotoxic activity testing is 12 hours. For example... Figure 5 As shown in C, the cytotoxic activity differs from that in DMEM (IC). 50 JZTX-66 (>50 μM) exhibited strong cytotoxic activity in calcium-free DMEM (IC50-50 μM). 50 =1.269 μM), with Ca 2+With increasing concentration, its activity gradually decreased, exhibiting a concentration-dependent effect, indicating that the cytotoxic activity of JZTX-66 was affected by the Ca in the culture medium. 2+ The influence of [unclear]. On the other hand, we will use 1640 medium (containing 0.4 mM Ca [unclear]). 2+ Based on ) and Ca 2+ The final concentrations were adjusted to 0.8 mM, 1.8 mM, and 2 mM to investigate the cytotoxic activity of JZTX-66 in these culture media, with the drug incubation time remaining at 12 h. Figure 5 D and Figure 5 E). Cytotoxicity results showed that JZTX-66 in the presence of 0.8 mM Ca 2+ The activity under the 1640 medium conditions was similar to that under the above conditions containing 0.8 mM Ca. 2+ The results were essentially consistent under calcium-free DMEM culture conditions; with the increase of Ca... 2+ With increasing concentration, the cytotoxic activity of the peptide is greatly reduced. However, the concentration of 1.8 mM or even 2 mM Ca... 2+ Under the 1640 medium conditions, the cytotoxic activity of the peptide remained similar to that of DMEM (Ca). 2+ The significant difference in activity at a concentration of 2 mM indicates that Ca... 2+ Although it significantly affected the cytotoxic activity of JZTX-66, other cations in the culture medium may also have contributed to its influence (see [link to article]). Figure 5 F).
[0033] 6. JZTX-66 induces apoptosis.
[0034] To investigate the effect of JZTX-66 on apoptosis of B16-F10 cells, we used flow cytometry to detect the proportion of apoptotic cells at each stage after treating B16-F10 cells with different concentrations (0 μM, 0.625 μM, 1.25 μM, 2.5 μM and 5 μM) of JZTX-66 for 24 h. Figure 6 This is a flow cytometry analysis (using Annexin V and PI double staining) of the effect of JZTX-66 on apoptosis in B16-F10 cells. In the figure, Q1 represents necrotic cells, Q2 represents late apoptotic cells, Q3 represents early apoptotic cells, and Q4 represents normal cells. The results showed that, similar to the control group, cells in each treatment group were mainly concentrated in the Q4 region. The late apoptotic cells in the 0.625 μM and 1.25 μM treatment groups were mainly below 10%, suggesting that JZTX-66 had no significant effect on apoptosis of B16-F10 cells at low concentrations. However, with increasing concentrations, the late apoptotic cells in the 2.5 μM and 5 μM treatment groups were 12.8% and 30.5%, respectively, indicating that apoptosis occurred in B16-F10 cells at these and higher concentrations.
[0035] 7. Analysis of the inhibitory activity of JZTX-66 on cell proliferation.
[0036] Based on our preliminary research plan, we adopted a method lower than IC 50 Cell colony formation assays were performed using different drug concentrations to assess the drug's effect on cell proliferation. Therefore, the optimal concentrations of JZTX-66 for cell colony formation were determined to be 1.25 µM, 0.625 µM, and 0.313 µM. Figure 7 As shown in Figure A, after 12 days of drug treatment, crystal violet staining revealed clearly visible blue-purple cell colonies in the control group, with a relatively large number of colonies. The cell colonies in the 1.25 µM and 0.625 µM JZTX-66 treatment groups were significantly smaller in both number and size than those in the control group, showing statistically significant differences. No significant difference was observed in the 0.313 µM JZTX-66 treatment group. Figure 7 B). The above results indicate that JZTX-66 has an inhibitory effect on the proliferation of B16-F10 cells. Secondly, cell proliferation is often inseparable from the cell cycle. Colony formation experiments showed that JZTX-66 can inhibit cell proliferation at low concentrations. To investigate the effect of low concentrations of JZTX-66 on the cell cycle of B16-F10 cells, we examined the cell cycle stage distribution of B16-F10 cells after 24 h of treatment with different concentrations (0 μM, 0.313 μM, 0.625 μM, and 1.25 μM) of JZTX-66. (See the DNA content distribution histogram). Figure 8 A) and cell cycle distribution bar chart ( Figure 8 As shown in B), the first peak of the DNA content distribution histogram represents the G0 / G1 phase, and no obvious peak representing apoptosis (sub-G1 phase) appears to the left of the G0 / G1 phase, indicating that no apoptosis occurred in B16-F10 cells treated with JZTX-66 at this low concentration, consistent with the flow cytometry results of apoptosis mentioned above. The second broad peak represents the S phase; the third peak represents the G2 / M phase. The ratios of each treatment phase show that, compared with the control group and the drug-treated group, the peak value of the S phase in the drug-treated group gradually increased (Control: 22.63%, 0.313 μM JZTX-66 treatment group: 22.95%, 0.625 μM JZTX-66 treatment group: 32.25%, and 1.25 μM JZTX-66 treatment group: 43.01%), that is, the number of cells in the S phase increased, indicating that cell S phase arrest occurred in B16-F10 cells after 24 h of JZTX-66 treatment.
[0037] 8. JZTX-66 inhibits cell migration and invasion.
[0038] Previous results indicated that low concentrations of anticancer peptides significantly inhibited melanoma cell metastasis. Therefore, in this study, we also investigated the effects of JZTX-66 on melanoma cell migration and invasion. Three concentrations (1.25 μM, 0.625 μM, and 0.313 μM) were selected (below IC50). 50 The concentration used to test the effects of JZTX-66 on cell migration and invasion. Figure 9 A and Figure 9 As shown in Figure C, compared with the control, the 1.25 μM and 0.625 μM treatment groups significantly inhibited the migration of B16-F10 cells, showing statistically significant differences, while the 0.313 μM treatment group showed no significant difference. Figure 9 B and Figure 9 As shown in Figure D, compared with the control, all three concentrations significantly inhibited the invasion of B16-F10 cells. In the invasion experiment, cells need to secrete matrix metalloproteinases and other substances to degrade matrix gel beforehand. In addition to inhibiting cell migration, JZTX-66 may also affect the secretion of cellular enzymes, which will further affect the cell invasion ability, possibly resulting in JZTX-66 having a stronger inhibitory activity on cell invasion than on migration.
[0039] 9. Analysis of the cell membrane disruption effect of JZTX-66.
[0040] Numerous studies have shown that membrane disruption is a major mechanism by which anticancer peptides and their analogues induce cell death, and this mechanism of action is rapid. The effects of JZTX-66 on the cell membrane were observed using scanning electron microscopy (SEM), such as... Figure 10 As shown in Figure A, unlike the intact cell membranes of the control group, the cell membranes treated with the peptide exhibited perforations, indicating that the cell membranes were damaged. Trypan blue can only penetrate the incomplete cell membranes to stain the cells blue. The cells treated with JZTX-66 were stained blue, indicating that the peptide disrupted the integrity of the cell membrane. Figure 10 B). SYTO9 is a nucleic acid dye that is permeable to cell membranes, staining both intact and damaged cells green, while PI can only permeate damaged cells, thus staining their nuclei red. For example... Figure 10 As shown in Figure C, the SYTO9 / PI double staining experiment showed that most cells were stained red after JZTX-66 treatment, further demonstrating the disruptive effect of the peptide on the cell membrane.
[0041] 10. Analysis of the therapeutic effect of EDTA-assisted JZTX-66 on tumors in mice.
[0042] Eight groups were established using 8 mg / mL EDTA as an adjuvant: control group, EDTA group, 100 μg JZTX-66 treatment group, 100 μg JZTX-66 + EDTA treatment group, 50 μg JZTX-66 treatment group, 50 μg JZTX-66 + EDTA treatment group, 25 μg JZTX-66 treatment group, and 25 μg JZTX-66 + EDTA treatment group. The intratumoral injection volume was 50 μL, administered once daily for three consecutive days, with the first injection designated as Day 0. Tumor volume and mouse weight were measured every two days. Figure 11 A). For example Figure 11 B and Figure 11 As shown in Figure C, the tumor volume in both the control and EDTA groups showed a gradual increasing trend. There was no significant difference in tumor volume between the 100 μg, 50 μg, and 25 μg JZTX-66 treatment groups and the control group. However, the tumors in mice treated with 100 μg JZTX-66 + EDTA, 50 μg JZTX-66 + EDTA, and 25 μg JZTX-66 + EDTA were significantly reduced, and some even showed signs of healing with scab regression. This effect was concentration-dependent. Compared with the control group, there was no significant change in body weight in any of the treatment groups, indicating that JZTX-66 treatment had no significant effect on the health of mice. Figure 11 D).
[0043] Research Methods 1. Preparation and amino acid sequence determination of natural anticancer peptide JZTX-66.
[0044] (1) Obtaining crude venom: Use tweezers to fix an adult female tarantula, extend its two chelicerae, and tightly grasp the opening of a 15 mL centrifuge tube. Then turn on the electric stimulation venom collection device, dip the two wires in a little physiological saline, and use the current to stimulate the two chelicerae to expel venom. After all the venom is collected, place it in a freeze dryer to freeze dry and obtain freeze-dried powder.
[0045] (2) Separation and purification: The lyophilized powder was dissolved in Q water to prepare a 10 mg / mL solution. After complete dissolution and centrifugation, the solution was filtered through a 0.22 μM filter and then separated and purified by high performance reversed-phase liquid chromatography (RP-HPLC) using a Hanbang semi-preparative chromatograph. The acetonitrile elution gradient was 10%~60%, and the mobile phase flow rate was 3 mL / min. All components were collected. All components were lyophilized and stored at -20 ℃. Then, the component containing JZTX-66 was further purified by a Waters 2695 analytical chromatograph with an acetonitrile elution gradient of 20%~30% and a mobile phase flow rate of 1 mL / min. The target elution peak was collected for mass spectrometry identification and subsequent experiments.
[0046] (3) In this study, the Edman degradation method and the in-solution enzymatic digestion-desalting-LC-MS / MS method were combined to determine the amino acid sequence of JZTX-66. The primary structure of the target peptide was determined to be: GDDGTCILKGDHCHGTCDCCGWTTTCRKSKSAGGKICKSEGSSISAFNAIAKGVAAMKKAKCKHKS.
[0047] 2. Prokaryotic expression of the anticancer peptide JZTX-66.
[0048] (1) Based on the amino acid sequence information of JZTX-66, its corresponding base sequence was optimized according to the codon preference of Escherichia coli (E. coli). pET 32a was selected as the prokaryotic expression vector, and the cloning site was Kpn I / Xho I. This vector has one Trx tag (to increase the expression of soluble proteins) and two 6×His tags (to facilitate affinity purification of fusion proteins). In this study, a TEV protease recognition sequence was inserted between the Kpn I site and the target sequence. Its amino acid sequence is ENLYFQG. The TEV enzyme will cut between Q and G. Since the first amino acid of the target sequence is G, only one G was retained during construction. This vector was completed by Sangon Biotech (Shanghai) Co., Ltd. Finally, the plasmid and glycerol bacteria constructed by the company were stored at -20 ℃ for later use.
[0049] (2) The plasmid JZTX-66-pET 32a constructed by the company was transformed into BL21 competent cells and expressed in the Escherichia coli prokaryotic system. The fusion protein obtained by expression was identified by SDS-PAGE, and after enzyme digestion, it was separated and purified by chromatography and mass spectrometry. Finally, the target sample was collected for activity identification, and the expression of the target protein JZTX-66 was confirmed to be successful.
[0050] 3. Assay of JZTX-66 cytotoxic activity.
[0051] Cytotoxic activity analysis of JZTX-66 against multiple cell lines: Cells in good condition were digested with trypsin and then subjected to a 2×10⁻⁶ ion exchange rate. 3 After seeding the cells in 96-well plates and culturing for 24 h, drug treatment was performed by serially diluting the drug twofold into serum-free 1640 medium and incubating for 24 h. Then, 10 μL of CCK-8 solution was added to each well and incubated for 1–4 h. The absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated as 1 – (drug OD – blank group OD) / (control group OD – blank group OD) × 100%. The IC50 of the drug was calculated using GraphPad Prism 5.0 software. 50 Values and graphs.
[0052] 4. The cytotoxic activity of JZTX-66 is affected by Ca. 2+ The influence of metal cations, etc.
[0053] We found that JZTX-66 exhibits varying toxicity to different cell types, such as B16-F10, 4T1, T24, and HTC-116 cells, with different IC50 values. 50 The values were between 2 and 6 μM, while the IC50 values for cells such as HeLa, A549, L-929, and HUVEC were significantly higher. 50 The values were all greater than 50 μM, prompting us to consider cell culture conditions. Comparison revealed that cell lines with weak JZTX-66 activity were all cultured in DMEM, while those with strong activity were all cultured in 1640 medium. To further verify the effect of culture medium on peptide activity, we conducted activity tests on selected cells using different culture medium components, ultimately determining that the cytotoxic activity of JZTX-66 is affected by Ca2+. 2+ The influence of metal cations, etc.
[0054] 5. JZTX-66 induces apoptosis.
[0055] A suitable amount of healthy B16-F10 cells were seeded into 6-well plates. After treatment with different concentrations of JZTX-66 for 24 h, the B16-F10 cells were digested with EDTA-free trypsin (the supernatant and digested cells were combined). The cells were centrifuged at 500 g for 10 min, and the supernatant was discarded. 5 mL of PBS was added and mixed, and the mixture was centrifuged at 400 g for 10 min. 500 μL of the mixture was retained and centrifuged again at 400 g for 10 min. The supernatant was carefully discarded, and 50 μL of the mixture was retained and placed on ice. 4× binding buffer was diluted to 1× with PBS. After removing any remaining PBS from the centrifuge tubes, 100 μL of 1× binding buffer was added to each tube. The cells were resuspended thoroughly by pipetting. Under light-protected conditions, 5 μL each of Annexin V and PI dyes were added and gently mixed with a pipette. After incubating at room temperature in the dark for 15 min, add 300 μL of 1× binding buffer and mix well. Then, transfer the cell suspension to a 5 mL flow cytometer in the dark and analyze it on a flow cytometer within 1 h.
[0056] The Annexin V-FITC / PI apoptosis detection kit uses FITC-labeled Annexin V as a probe. FITC has a maximum excitation wavelength of 488 nm and a maximum emission wavelength of 525 nm; FITC's green fluorescence is detected in the FL1 channel. The PI-DNA complex has a maximum excitation wavelength of 535 nm and a maximum emission wavelength of 615 nm; PI's red fluorescence is detected in the FL2 or FL3 channel. Software analysis was used to create a two-color scatter plot, with FITC on the x-axis and PI on the y-axis.
[0057] 6. JZTX-66 inhibits cell proliferation.
[0058] Cell colony formation assay: B16-F10 cells were digested with 0.25% trypsin and seeded into 12-well plates at a density of approximately 50 cells / well. After culturing for 36 h, drug treatment was performed. The control group only had its complete culture medium replaced, while the treatment groups had complete culture medium added to prepare JZTX-66 at final concentrations of 1.25 μM, 0.625 μM, and 0.313 μM, with 3 replicates per group. After 5 days of treatment, the drug was added again, and the cells were incubated for a total of 12 days. When obvious colonies were observed to appear visually, the old culture medium was gently discarded, and the cells were washed with PBS. Then, an appropriate amount of 2.5% crystal violet solution was added to each well for staining for 3 min. Finally, the cells were washed 3 times with PBS, air-dried, and the colony formation in each well was photographed and the colony count was recorded.
[0059] Cell cycle detection: A suitable amount of healthy B16-F10 cells were seeded in 6-well plates and treated with different concentrations of JZTX-66 for 24 h. The B16-F10 cells were then digested with trypsin, centrifuged at 600 g for 10 min, and the supernatant was discarded. PBS was added, and the cells were washed by centrifugation at 400 g. The cell pellet was gently mixed with 500 μL of PBS, and the cell suspension was slowly added to 4 mL of pre-chilled 95% ethanol solution. The mixture was then incubated at 4 ℃ for 12–24 h. After the fixation time, the cells were centrifuged at 400 g for 10 min, the supernatant was removed, and the cells were placed on ice. 50 µL of RNase stock solution (100 µg / mL) was added to treat the cells, followed by 200 µL of PI. The cells were incubated at room temperature in the dark for 30 min before being analyzed by flow cytometry.
[0060] 7. JZTX-66 inhibits cell migration and invasion.
[0061] B16-F10 cells were digested and resuspended in serum-free 1640 medium for later use. Cell migration ability was tested using Transwell plates. Control group: 600 μL of serum-containing 1640 medium was added to each well in the lower chamber, and 100 μL of cell suspension was added to each well in the upper chamber. Drug treatment groups (1.25 μM, 0.625 μM, 0.313 μM): 600 μL of serum-containing 1640 medium (containing the drug) was added to each well in the lower chamber, and 100 μL of cell suspension (containing the drug) was added to each well in the upper chamber. The lower chamber was added first, followed by the upper chamber, ensuring no air bubbles formed in either chamber. PBS was added to the surrounding non-experimental wells, and the plates were incubated at 37 ℃ for 24 h. After 24 h, the liquid in the upper chamber was gently aspirated with a pipette tip, and each upper chamber was quickly transferred to a new 24-well plate for cell fixation (wells were pre-filled with 100% methanol solution). After 8 min of fixation, the cells in the upper chamber wells were wiped away with a moistened cotton swab. The chambers were then inverted and air-dried, followed by staining in wells containing 2.5% crystal violet solution. After 3-5 minutes, the upper chamber was removed, washed several times in PBS, and air-dried. Finally, the chambers were observed under a microscope, and the results were photographed and recorded. The procedure for the invasion experiment is basically the same as that for the migration experiment, except that a layer of matrix gel is pre-laid inside the chamber to simulate the ECM environment before the experiment.
[0062] Characterization of JZTX-66's disruption of cell membranes.
[0063] Scanning electron microscopy (SEM) analysis: Well-preserved B16-F10 samples were digested and seeded at an appropriate density in pre-laid sterile slides in six-well plates. After 24 h of incubation, drug treatment was performed. Different final concentrations (10 μM, 5 μM) of JZTX-66 were prepared using serum-free 1640 medium and treated for 1 h. After washing with PBS, SEM fixative was added and fixed at room temperature for 2 h, then transferred to 4 ℃ for storage. The fixed slides were rinsed three times with PBS for 15 min each time. The slides were then fixed with 1% osmium tetroxide at room temperature in the dark for 1–2 h. After rinsing three times with PBS for 15 min each time, the samples were dehydrated sequentially with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol for 15 min each, followed by further dehydration with isoamyl acetate for 15 min. The dehydrated samples were dried in a critical point desiccator, sputter-coated with gold, and then observed and imaged under a scanning electron microscope.
[0064] Trypan blue staining: Trypan blue stains dead cells while rejecting live cells, serving as an indicator of cell viability. Cells were digested with 0.25% trypsin and seeded in 12-well plates at a density of 1000-2000 cells / well. After culturing for 24 h, cells were treated with serum-free 1640 medium containing 10 μM and 5 μM JZTX-66 for 1 h. The medium was then discarded, and pre-prepared 0.4% trypan blue dye was added for staining for 3-5 min. Cells were then washed three times with PBS, and images of both live and dead cells were recorded.
[0065] SYTO9 / PI staining: B16-F10 cells were seeded at an appropriate density in 48-well plates and allowed to adhere for 24 h. After the culture medium was discarded, serum-free 1640 medium containing JZTX-66 was added and incubated for 1 h. After the supernatant was discarded, serum-free 1640 medium containing SYTO9 / PI dye was added and incubated for 10 min. The cells were then observed under a fluorescence microscope, photographed, and the staining results were recorded.
[0066] 9. In vivo antitumor activity study of anticancer peptide JZTX-66.
[0067] To further investigate the in vivo antitumor activity of JZTX-66, we constructed a B16-F10 mouse tumor-bearing model. Male BALB / c mice, aged 4-6 weeks and weighing 18-20 g, were purchased from Changsha Silex Co., Ltd. The purchased mice were placed in the animal room for 1-2 days to acclimatize, followed by hair removal on the lower back area. The next day, B16-F10 cells were digested, resuspended in PBS, and cultured at 2×10⁻⁶ cells / mL. 6 The tumors were injected into the backs of mice at a specific density. The tumors were allowed to grow to 100-150 mm. 3 At that time, drug administration was performed. Considering that the activity of JZTX-66 is affected by Ca... 2+ Given the characteristics of the influence of metal cations, the proposed method is to use EDTA to dechelate Ca in tumor cells. 2 +The tumor-suppressive effect of peptides was analyzed by intratumoral injection using metal cations. Eight groups were established using 8 mg / mL EDTA as an adjuvant: control group, EDTA group, 100 μg JZTX-66 treatment group, 100 μg JZTX-66 + EDTA treatment group, 50 μg JZTX-66 treatment group, 50 μg JZTX-66 + EDTA treatment group, 25 μg JZTX-66 treatment group, and 25 μg JZTX-66 + EDTA treatment group. The intratumoral injection volume was 50 μL, administered once daily for three consecutive days, with the first injection designated as Day 0. Tumor volume and mouse weight were measured every two days. After the monitoring period, mice were euthanized, and the heart, liver, spleen, lungs, kidneys, and tumor tissues were dissected and placed in 4% paraformaldehyde for subsequent HE and TUNEL staining. This animal experiment has been approved by the Ethics Committee of Hunan Normal University.
Claims
1. An anticancer peptide, characterized in that, The polypeptide is: the application of the anticancer peptide JZTX-66 derived from the spider *Gymnodon dactylus* in the preparation of an anticancer drug that inhibits melanoma B16-F10 cells. The amino acid sequence of the anticancer peptide JZTX-66 is GDDGTCILKGDHCHGTCDCCGWTTTCRKSKSAGGKICKSEGSSISAFNAIAKGVAAMKKAKCKHKS.
2. The anticancer peptide according to claim 1, characterized in that: Ca 2+ Metal cations can inhibit the cytotoxic activity of JZTX-66, and this inhibition can be reversed by the cationic chelator EDTA. The anticancer activity of JZTX-66 is affected by Ca. 2+ Regulation by metal cations.
3. The anticancer peptide according to claims 1 and 2, characterized in that: In low Ca 2+ Under conditions of metal cations, it can induce apoptosis through multiple modes of action, namely high concentration of peptides or direct destruction of cell membranes leading to cell death; low concentration of peptides has a potent inhibitory effect on the proliferation, migration and invasion of B16-F10 cells, which are melanoma cells.
4. According to the anticancer peptides described in claims 1, 2, and 3, JZTX-66 can be developed into a Ca2+-dependent anticancer peptide. 2+ Safe and highly effective anticancer drugs containing metal cations, including melanoma, breast cancer, lung cancer, cervical cancer, colon cancer, ovarian cancer, and leukemia.