Application of scorpion venom polypeptide in preparation of medicine for preventing and / or treating lung adenocarcinoma

Abstract

The invention discloses application of scorpion venom polypeptide in preparation of a medicine for preventing and / or treating lung adenocarcinoma. The invention discloses application of scorpion venom polypeptide S6540 in preparation of a medicine for preventing and / or treating lung adenocarcinoma. The scorpion venom polypeptide S6540 is found to have no obvious toxic or side effect, can inhibit proliferation and migration of lung adenocarcinoma cells, can destroy cytoskeletons, can directly penetrate through cell membranes to enter cells and cause mitochondrial dysfunction to cause tumor cell cycle arrest and induce apoptosis to play an anti-tumor role, and can be used for preparing anti-tumor drugs for treating lung adenocarcinoma. The scorpion venom polypeptide S6540 is not influenced by tumor cell drug resistance caused by mutation, the drug resistance is effectively reduced, and the scorpion venom polypeptide S6540 can also inhibit angiogenesis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the use of a scorpion venom polypeptide in the preparation of a drug for preventing and / or treating lung adenocarcinoma. Background Art

[0002] Lung adenocarcinoma is a malignant tumor originating from glandular epithelial cells in the lungs and is the most common type of non-small cell lung cancer. The annual incidence of lung adenocarcinoma in China is approximately 15 - 20 per 100,000, and the mortality rate ranks relatively high among all malignant tumors. The clinical manifestations of lung adenocarcinoma are usually atypical, and early diagnosis is difficult. Many patients are already in the advanced stage at the time of diagnosis, resulting in limited treatment effects. The current main treatment methods for lung adenocarcinoma include surgical resection, radiotherapy, chemotherapy, and targeted therapy, etc. However, surgery requires lobectomy or segmentectomy of the lung, with relatively high risks and a long postoperative recovery period; while chemotherapy and radiotherapy, due to their non-selective effects, not only kill cancer cells but may also damage normal tissues, leading to serious side effects and affecting the quality of life of patients; although targeted therapy has a certain degree of selectivity, it may still develop drug resistance and has limited effects on certain specific mutant types of lung adenocarcinoma. Therefore, for the treatment of lung adenocarcinoma, there is an urgent need to develop a new treatment plan with fewer side effects, higher selectivity for cancer cells, and the ability to effectively overcome drug resistance to improve the prognosis of lung adenocarcinoma patients. Summary of the Invention

[0003] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide the use of scorpion venom polypeptide S6540 in a drug for inhibiting the growth of lung adenocarcinoma, and to develop and seek new, highly effective and low-toxic drugs for the treatment of lung adenocarcinoma.

[0004] Scorpion venom is a mixture produced by protein (enzymes and peptides) and non-protein (inorganic salts, lipids, nucleotides, free amino acids, and water) by the venom gland for defense and prey capture. It has been reported to have important effects such as antibacterial, antiviral, blood pressure lowering, and blood sugar lowering. Due to its small molecular weight and easy absorption, it can be widely used in the development of health products and drugs. Anticancer peptides (ACPs) are mainly derived from natural and artificial production, and exert their antitumor effects by targeting cell membranes rather than specific antibodies. Anticancer peptides can selectively bind to cancer cells, causing membrane lysis and the death of cancer cells. In addition to inducing membrane lysis, anticancer peptides can also exert toxicity on tumors by affecting the functions of different cell structures, thereby interfering with various cellular processes such as apoptosis, cell cycle distribution, and autophagy.

[0005] S6540 is a cationic polypeptide identified from the venom of the scorpion Superstitionia donensis, and its biological activity has not been reported. The present invention discovers that scorpion venom polypeptide S6540 has the effect of inhibiting the proliferation of lung adenocarcinoma cells.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides the application of scorpion venom polypeptide S6540 in the preparation of a drug for preventing and / or treating lung adenocarcinoma.

[0008] The present invention also provides the application of scorpion venom polypeptide S6540 in the preparation of a health product.

[0009] The present invention also provides the application of scorpion venom polypeptide S6540 in the preparation of a preparation for inhibiting the proliferation of lung adenocarcinoma cells.

[0010] The present invention also provides the application of scorpion venom polypeptide S6540 in the preparation of a preparation for inducing apoptosis of lung adenocarcinoma cells.

[0011] The present invention also provides the application of scorpion venom polypeptide S6540 in the preparation of a preparation for inhibiting the migration and / or angiogenesis of lung adenocarcinoma cells.

[0012] The present invention also provides the application of scorpion venom polypeptide S6540 in the preparation of a preparation for destroying the membrane stability of lung adenocarcinoma cells or mitochondrial membrane stability.

[0013] The present invention also provides the application of scorpion venom polypeptide S6540 in the preparation of a preparation for inducing mitochondrial dysfunction in lung adenocarcinoma cells.

[0014] Preferably, the lung adenocarcinoma cells are A549 cells.

[0015] The present invention also provides a composition, which comprises scorpion venom polypeptide S6540.

[0016] Furthermore, the composition further comprises a pharmaceutically or food-acceptable carrier.

[0017] Furthermore, the amino acid sequence of the scorpion venom polypeptide S6540 is as shown in SEQ ID NO.1, which is a polypeptide composed of 18 amino acids, and its amino acid sequence is: GFWGNVWEGIKSVGKNLL.

[0018] The present invention provides that the scorpion venom polypeptide S6540 can be prepared by methods known to those skilled in the art (such as solid-phase synthesis methods), and can be separated and purified by separation and purification methods known in the art (such as high performance liquid chromatography).

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention discovers the application of scorpion venom polypeptide S6540 in the preparation of drugs for preventing and / or treating lung adenocarcinoma. The present invention uses human lung adenocarcinoma cells for in vitro tumor cell experiments, and verifies the therapeutic effect through a mouse tumor-bearing model. The results show that scorpion venom polypeptide S6540 has good anti-tumor effects and no obvious toxic side effects, and can inhibit the proliferation and migration of lung adenocarcinoma cells. Scorpion venom polypeptide S6540 can disrupt the cytoskeleton, directly penetrate the cell membrane into the cell and cause mitochondrial dysfunction, leading to cell cycle arrest of tumor cells and inducing apoptosis, without being affected by tumor cell drug resistance caused by mutations, effectively reducing drug resistance. Scorpion venom polypeptide S6540 can also inhibit angiogenesis. The scorpion venom polypeptide S6540 has the beneficial characteristics of simple structure, convenient artificial synthesis, strong activity and selectivity for normal cells. Scorpion venom polypeptide S6540 can not only be used alone as a biotherapeutic drug for anti-tumor, but also has the potential to combine with other treatment methods to inhibit tumors. Description of the Drawings

[0021] Figure 1 .The purity and identification of the synthesized S6540 were detected by HPLC (A) and MALDI-TOF MS; (B) respectively.

[0022] Figure 2 .A: Inhibitory effect of S6540 on the proliferation of A549 cells; B: LDH level released by S6540-induced A549 cells; C-D: Cytotoxicity of S6540 on A549 cells in the presence of Z-DEVD-FMK (DEVD, 40 μM), Z-VAD-FMK (VAD, 40 μM), disulfiram (DSF, 10 μM) and N-acetyl-L-cysteine (NAC, 2 mM).

[0023] Figure 3 .A: Flow cytometry analysis of calcein released by A549 cells; B: Flow cytometry analysis of calcein released by A549 cells after adding CoCl2. C: Flow cytometry analysis of excessive accumulation of oxygen free radicals in S6540-induced A549 cells.

[0024] Figure 4 .A: Flow cytometry analysis of the apoptosis level of A549 cells induced by S6540 or cisplatin; B: Screening of the catalytic effect of S6540 on substrates using different caspase chromogenic substrates; C: Western blots analysis of the effects of S6540 or cisplatin on apoptosis-related signaling pathway proteins in A549 cells; D: Statistical analysis of Figure B.

[0025] Figure 5. A: Scratch assay was used to analyze the effect of S6540 at different doses on the migration ability of A549 cells; B: Transwell chamber assay was used to analyze the effect of S6540 on the invasion ability of A549 cells.

[0026] Figure 6 . A: Microscopic observation was used to analyze the effect of S6540 at different doses on the tube formation level of human umbilical vein endothelial cells (HUVEC); B: Transwell chamber assay was used to analyze the effect of S6540 on the migration ability of HUVEC cells.

[0027] Figure 7 . A: The inhibitory effect of S6540 on the subcutaneous tumor growth of nude mice inoculated with A549 cells; B: Changes in tumor volume; C: The effect of S6540 on tumor weight; D: The effect of S6540 on the body weight of tumor-bearing nude mice; E: Pathological sections were used to evaluate the effect of S6540 on inducing tumor apoptosis and necrosis.

[0028] Figure 8 . The effect of S6540 on the liver and kidney functions of normal nude mice. A: Aspartate aminotransferase; B: Creatinine; C: Alanine aminotransferase; D: Blood urea nitrogen. Detailed implementation mode

[0029] To understand the present invention more clearly, the present invention will be further described with reference to the following embodiments and accompanying drawings. The embodiments are only for explanation and do not limit the present invention in any way. In the embodiments, all the original reagent materials are commercially available, and the experimental methods without specific conditions are the conventional methods and conventional conditions well known in the art, or the conditions recommended by the instrument manufacturer.

[0030] Synthetic scorpion venom polypeptide S6540, whose amino acid sequence is: GFWGNVWEGIKSVGKNLL, as shown in SEQ ID NO.1. The purity and identification of the synthesized S6540 were detected by HPLC (A) and MALDI-TOF MS (B) respectively, as Figure 1 shown. The following experiments were carried out using the synthesized scorpion venom polypeptide S6540.

[0031] Example 1: Inhibitory effect of scorpion venom polypeptide S6540 on the proliferation activity of lung adenocarcinoma cells

[0032] The CCK8 method was used to detect the effect of scorpion venom polypeptide S6540 on inhibiting the proliferation ability of A549 cells in vitro. As Figure 2 shown, with the increase of concentration, scorpion venom polypeptide S6540 inhibited A549 cells, and the half-maximal inhibitory concentration IC50 was 9.84 μmol / L.

[0033] Example 2: Interference of scorpion venom polypeptide S6540 with the mitochondrial membrane stability of lung adenocarcinoma cells

[0034] Flow cytometry was used to analyze the calcein released by A549 cells after the action of scorpion venom polypeptide S6540. As Figure 3 shown in Figures 3A and 3B, with the increase of concentration, the accumulated fluorescence of A549 cells gradually decreased, indicating that scorpion venom polypeptide S6540 caused the change of cell membrane permeability. After adding the calcein quencher CoCl2 on this basis, it was observed that the fluorescence value retained in mitochondria also decreased in a concentration-dependent manner, indicating that the mitochondrial membrane of A549 cells was also damaged.

[0035] Flow cytometry was used to analyze the cellular oxygen free radicals of A549 cells after the action of scorpion venom polypeptide S6540. As Figure 3 shown in Figure 3C, with the increase of concentration, the fluorescence of oxygen free radicals gradually increased, indicating that the polypeptide caused the excessive accumulation of intracellular oxygen free radicals.

[0036] Example 3: Scorpion venom polypeptide S6540 interferes with the apoptosis of lung adenocarcinoma cells

[0037] Flow cytometry was used to analyze the apoptosis rate of A549 cells after the action of scorpion venom polypeptide S6540. As Figure 4 shown, with the increase of concentration, the apoptosis rate gradually increased, indicating that scorpion venom polypeptide S6540 caused the excessive apoptosis of A549 cells; Western blot experiments showed that S6540 could induce cell apoptosis through the caspase-independent pathway.

[0038] Example 4: Scorpion venom polypeptide S6540 inhibits the migration and invasion of lung adenocarcinoma cells

[0039] Cell scratch, Transwell cell migration and other experiments were used to analyze the cell migration and invasion ability of A549 cells after the action of scorpion venom polypeptide S6540. As Figure 5 shown, the results showed that at non-lethal concentrations, with the increase of concentration, the migration and invasion ability of A549 cells gradually weakened; the results of F-actin staining experiments showed that S6540 affected the cell migration ability by destroying the normal structure of actin microfilaments.

[0040] Example 5: Scorpion venom polypeptide S6540 inhibits angiogenesis

[0041] Tube formation experiments were used to analyze the inhibitory effect of polypeptide S6540 on HUVEC cells. As Figure 6 shown, the results showed that at non-lethal concentrations, with the increase of concentration, the newly formed tubules gradually decreased; the results of Transwell cell migration experiments showed that S6540 could inhibit the migration of HUVEC.

[0042] Example 6: The role of scorpion venom polypeptide S6540 in the treatment of lung adenocarcinoma

[0043] The in vivo anti-cancer ability of S6540 was evaluated using a xenograft model, as Figure 7 shown, and the results showed that S6540 effectively reduced the tumor volume and weight.

[0044] Example 7: Effects of scorpion venom polypeptide S6540 on the liver and kidney functions of normal nude mice

[0045] Figure 8 Detection of the effects of scorpion venom polypeptide S6540 on the liver and kidney functions of normal nude mice indicated that scorpion venom polypeptide S6540 was of low toxicity.

[0046] In summary, scorpion venom polypeptide S6540 exerts anti-cancer effects in vitro and in vivo by targeting lung adenocarcinoma cells, interfering with the mitochondrial membrane stability of tumor cells, and inducing apoptosis through the caspase-independent pathway.

[0047] Obviously, the above examples are only for clear illustration and not a limitation on the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description.

Claims

1. Use of scorpion venom polypeptide S6540 in the preparation of a drug for preventing and / or treating lung adenocarcinoma, characterized in that: The amino acid sequence of the scorpion venom polypeptide S6540 is shown in SEQ ID NO.

1.

2. Application of scorpion venom polypeptide S6540 in the preparation of health products, characterized in that: The amino acid sequence of the scorpion venom polypeptide S6540 is shown in SEQ ID NO.

1.

3. Use of scorpion venom polypeptide S6540 in the preparation of a preparation for inhibiting the proliferation of lung adenocarcinoma cells, characterized in that: The amino acid sequence of the scorpion venom polypeptide S6540 is shown in SEQ ID NO.

1.

4. Use of scorpion venom polypeptide S6540 in the preparation of a preparation for inducing apoptosis of lung adenocarcinoma cells, characterized in that: The amino acid sequence of the scorpion venom polypeptide S6540 is shown in SEQ ID NO.

1.

5. Use of scorpion venom polypeptide S6540 in the preparation of a preparation for inhibiting lung adenocarcinoma cell migration and / or angiogenesis, characterized in that: The amino acid sequence of the scorpion venom polypeptide S6540 is shown in SEQ ID NO.

1.

6. Use of scorpion venom polypeptide S6540 in the preparation of a preparation for destroying the cell membrane stability of lung adenocarcinoma cells or the stability of mitochondrial membranes, characterized in that: The amino acid sequence of the scorpion venom polypeptide S6540 is shown in SEQ ID NO.

1.

7. Use of scorpion venom polypeptide S6540 in the preparation of a preparation for inducing mitochondrial dysfunction in lung adenocarcinoma cells, characterized in that: The amino acid sequence of the scorpion venom polypeptide S6540 is shown in SEQ ID NO.

1.

8. The use according to any one of claims 3 to 7, characterized in that: The lung adenocarcinoma cells are A549 cells.

9. A composition, characterized in that The composition comprises scorpion venom polypeptide S6540, and the amino acid sequence of scorpion venom polypeptide S6540 is shown in SEQ ID NO.

1.

10. The composition according to claim 9, characterized in that The composition also includes a pharmaceutically or food acceptable carrier.

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