Application of recombinant humanized III-type collagen in preparation of esophageal squamous cell carcinoma inhibitor

Abstract

The invention relates to application of recombinant humanized III-type collagen in preparation of an esophageal squamous cell carcinoma inhibitor. The invention discovers that the recombinant humanized III-type collagen can significantly inhibit the proliferation of esophageal squamous carcinoma cells and prevent the generation of malignant phenotypes of esophageal squamous carcinoma cells. Therefore, the invention can be used for preparing medicines for preventing and treating esophageal squamous carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of recombinant humanized type III collagen in the preparation of esophageal squamous cell carcinoma inhibitors. Background Art

[0002] Esophageal squamous cell carcinoma (ESCC) is the main pathological type of esophageal cancer. Its early symptoms are not obvious, and it is usually diagnosed in the middle and late stages with a poor prognosis. ESCC is clinically treated with chemotherapy drugs such as platinum, paclitaxel, and fluorouracil, or a combination of multiple chemotherapy drugs. Although these regimens can curb tumor development in a short period of time, they are very likely to produce drug resistance. Once the drug fails, the tumor will be difficult to control, ultimately leading to adverse consequences.

[0003] Recombinant humanized type III collagen (rhCOLIII) is a new type of biomaterial with multiple biological functions, such as promoting skin extracellular matrix regeneration and improving cell microenvironment. rhCOLIII is composed of 16 tandem repeats of the functional domain of human type III collagen α1 chain (rhCOLIIIα1) and has a flexible triple helix structure of 164.88°. Compared with collagen from animal sources, rhCOLIII has the advantages of low antigenicity, high bioactivity, high hydrophilicity and no viral risk. Studies have shown that rhCOLIII exhibits significant anti-tumor activity in the treatment of ovarian cancer and breast cancer, and can inhibit the proliferation, migration and invasion of tumor cells, promote tumor cell apoptosis and dormancy, and inhibit cell autophagy. Summary of the invention

[0004] The present invention finds that recombinant humanized type III collagen can significantly inhibit the proliferation of esophageal squamous cell carcinoma cells and can be used to prepare esophageal squamous cell carcinoma inhibitors.

[0005] Therefore, the present invention provides an application of recombinant humanized type III collagen in the preparation of an esophageal squamous cell carcinoma inhibitor, wherein the core sequence of the recombinant humanized type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0006] In one embodiment, the esophageal squamous cell carcinoma inhibitor can inhibit the growth of esophageal squamous cell carcinoma cells and / or induce cell morphology changes.

[0007] In one embodiment, the esophageal squamous cell carcinoma cells include esophageal squamous cell carcinoma cells KYSE410 and esophageal squamous cell carcinoma cells KYSE510.

[0008] The present invention also provides a pharmaceutical composition for inhibiting esophageal squamous cell carcinoma, which comprises recombinant humanized type III collagen or a structural analogue thereof, and the core sequence of the recombinant humanized type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0009] In one embodiment, the pharmaceutical composition further comprises other drugs helpful for the treatment of esophageal squamous cell carcinoma.

[0010] Preferably, the other drugs are selected from platinum-based drugs, paclitaxel, and fluorouracil drugs; more preferably, the other drugs include at least one of cisplatin, paclitaxel, and fluorouracil.

[0011] Preferably, the mass ratio of the recombinant humanized type III collagen and its structural analogue to the other drugs is 1-10:10-1.

[0012] In one embodiment, the inhibitor or the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier or excipient.

[0013] The inhibitor or the pharmaceutical composition can be prepared according to methods well known in the art. By combining the active compound with a pharmaceutically acceptable carrier or excipient, any dosage form suitable for human or animal use can be made. The content of the recombinant humanized type III collagen and its structural analogue in the pharmaceutical composition is usually 0.1-95% by weight.

[0014] The recombinant humanized type III collagen and its structural analogue in the form of a dosage unit can be administered once or multiple times at appropriate intervals every day. Conveniently, the preparation of the dosage unit contains 0.1 mg to 1000 mg, preferably 1 mg to 100 mg, such as 5-50 mg of the recombinant humanized type III collagen or its structural analogue. The optimal dosage for administration can be easily determined by professionals in the art and will vary depending on the specific compound used, the mode of administration, the strength of the preparation, the route of administration, and the progression of the disease condition. Additionally, factors related to the specific patient being treated, including the patient's age, weight, diet, and frequency of administration, will create the need to adjust the dosage.

[0015] The preparations include, for example, preparations suitable for oral administration (including sustained release or timed release), rectal, parenteral (including subcutaneous, intraperitoneal, intramuscular, intra-articular, and intravenous), transdermal, ophthalmic, topical, dermal, nasal, buccal, or intradermal administration.

[0016] The dosage form can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form can be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type and multiple emulsion), a suspension, an injection (including aqueous injection, powder injection and infusion), an eye drop, a nasal drop, a lotion, a liniment, etc.; the solid dosage form can be a tablet (including ordinary tablet, enteric-coated tablet, lozenge, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric-coated capsule), a powder, a granule, a powder, a pellet, a dropping pill, a suppository, a film, a patch, an aerosol, a spray, etc.; the semi-solid dosage form can be an ointment, a gel, a paste, etc. The dosage form can also be a liposome.

[0017] Examples of the preparations for oral administration are tablets, pills, hard / soft capsules, solutions, suspensions, emulsions, syrups, powders, granules, etc. These preparations may contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine) and lubricants (e.g., silica, talc, stearates and their magnesium or calcium salts, and / or polyethylene glycol). Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone, and if necessary, disintegrants such as starch, agarose, alginic acid or its sodium salt or azeotropic mixture, and / or adsorbents, colorants, flavoring agents and sweetening agents may also be included therein.

[0018] The inhibitor or pharmaceutical composition of the present invention can be administered orally or parenterally and is used in the general form of a pharmaceutical preparation, but is not limited thereto. Beneficial effects

[0019] The present invention provides the application of recombinant humanized type III collagen in the preparation of an esophageal squamous cell carcinoma inhibitor. In cell experiments, recombinant humanized type III collagen can inhibit the progression of esophageal squamous cell carcinoma by inhibiting the proliferation of esophageal squamous cell carcinoma cells and changing the malignant phenotype of the cells. The experimental results show that, evaluated by the CCK8 method, recombinant humanized type III collagen can dose-dependently inhibit the growth of esophageal squamous cell carcinoma cell lines KYSE410 and KYSE510. By observing the effects of different doses of recombinant humanized type III collagen on the morphology of KYSE410 and KYSE510, it can be seen that recombinant humanized type III collagen can dose-dependently inhibit the generation of the malignant phenotype of KYSE410 and KYSE510. Description of the drawings

[0020] Figure 1 It is a schematic diagram of the result that the recombinant humanized type III collagen of Example 1 can inhibit the growth of esophageal squamous cell carcinoma cell line KYSE-410.

[0021] Figure 2Schematic diagram of the result that the recombinant humanized type III collagen of Example 1 can inhibit the growth of esophageal squamous cell carcinoma cell line KYSE-510.

[0022] Figure 3 Schematic diagram of the result that the recombinant humanized type III collagen of Example 2 can inhibit the generation of malignant phenotypes of esophageal squamous cell carcinoma cell line.

[0023] Figure 4 Schematic diagram of the result that the recombinant humanized type III collagen of Example 3 can achieve a more effective growth inhibitory effect in esophageal squamous cell carcinoma cells compared with other types of tumor cells.

[0024] In each of the above figures, represents P <0.01; represents P <0.001; represents P <0.0001. Detailed implementation manners

[0025] The preferred examples of the invention will be described in detail below. The examples are given to better state the content of the invention, and the content of the invention is not limited to the examples. Non-essential improvements and adjustments to the implementation manners according to the content of the invention still fall within the scope of the invention.

[0026] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. For those not specified in the examples regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications.

[0027] The recombinant humanized type III collagen used in the experiment was purchased from Shanxi Jinbo Biopharmaceutical Co., Ltd., and the amino acid core sequence (Gly483-Pro512) was: GERGAPGFRGPAGPNGIPGEKGPAGERGAP (SEQ ID NO.1).

[0028] Example 1: Growth inhibitory effect of recombinant humanized type III collagen on esophageal squamous cell carcinoma cell line

[0029] Cell growth ability determination The recombinant humanized type III collagen was dissolved in RPMI 1640 medium containing 10% FBS to prepare a stock solution of 50 mg / mL, and then serially diluted 2-fold to obtain recombinant humanized type III collagen solutions with different concentrations. The KYSE-410 and KYSE-510 cell lines were respectively seeded at 3.5×10 3Inoculate cells at a density of cells / well into a 96-well plate. After the cells adhere to the wall, add recombinant humanized type III collagen at different concentrations (25 mg / mL, 50 mg / mL). After 48 hours, add 10 μL of CCK8 solution to each well. Incubate the 96-well plate at 37 °C for 1 h and then measure the absorbance value at a wavelength of 450 nm. Use GraphPad Prism software to plot a schematic diagram of the growth inhibition rate of recombinant humanized type III collagen at different concentrations compared to the control group.

[0030] Control attachment Figure 1 Attachment Figure 2 The results showed that recombinant humanized type III collagen could inhibit the growth of esophageal squamous cell carcinoma cell lines.

[0031] Example 2: Cell morphological changes Inoculate KYSE-410 and KYSE-510 cell lines according to the method in Example 1. After adding recombinant humanized type III collagen at different concentrations (25 mg / mL, 50 mg / mL) for 48 hours, observe the cell morphology with an electron microscope.

[0032] Control attachment Figure 3 The results showed that recombinant humanized type III collagen could inhibit the generation of malignant phenotypes of esophageal squamous cell carcinoma cell lines.

[0033] Example 3: Recombinant humanized type III collagen can achieve more effective tumor growth inhibition in esophageal squamous cell carcinoma cells than other types of tumor cells Inoculate KYSE510, A2780, and MDB231 cell lines into 96-well plates respectively. After the cells adhere to the wall, add recombinant humanized type III collagen at different concentrations (25 mg / mL, 50 mg / mL). After 48 hours, add 10 μL of CCK8 solution to each well. Incubate the 96-well plate at 37 °C for 1 h and then measure the absorbance value at a wavelength of 450 nm. Inoculate KYSE-510, A2780, and MDB231 cell lines according to the above method, add recombinant humanized type III collagen (25 mg / mL, 50 mg / mL), and after 48 hours, add 10 μL of CCK8 solution to each well. Incubate the 96-well plate at 37 °C for 1 h and then measure the absorbance value at a wavelength of 450 nm. Use GraphPad Prism software to plot a schematic diagram of the growth inhibition rate of recombinant humanized type III collagen at different concentrations compared to the control group.

[0034] Control attachment Figure 4 The results showed that recombinant humanized type III collagen can achieve more effective tumor growth inhibition in esophageal squamous cell carcinoma cells than other types of tumor cells.

[0035] Example 4: Recombinant humanized type III collagen was encapsulated in liposomes to prepare liposomal recombinant humanized type III collagen as a drug for treating esophageal squamous cell carcinoma.

[0036] Example 5: Recombinant humanized type III collagen was added to excipients in the proportion required for the preparation to make tablets.

[0037] Example 6: Recombinant humanized type III collagen was added to excipients in the proportion required for the preparation to make powders.

[0038] Example 7: The extract of recombinant humanized type III collagen was spray granulated, dried, and sized to obtain granules as a drug and health product for preventing and treating esophageal squamous cell carcinoma.

[0039] Example 8: Recombinant humanized type III collagen was dissolved in water, finely filtered, sealed, and sterilized to make injections.

[0040] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A use of recombinant humanized type III collagen in the preparation of an esophageal squamous cell carcinoma inhibitor, wherein the core sequence of the recombinant humanized type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

2. The use according to claim 1, characterized in that: The esophageal squamous cell carcinoma inhibitor can inhibit the growth of esophageal squamous cell carcinoma cells and / or induce cell morphology changes.

3. The use according to claim 2, characterized in that: The esophageal squamous cell carcinoma cells include esophageal squamous cell carcinoma cells KYSE410 and esophageal squamous cell carcinoma cells KYSE510.

4. The use according to any one of claims 1 to 3, characterized in that: The inhibitors are suitable for oral, rectal, parenteral, transdermal, ocular, topical, cutaneous, nasal, buccal or intradermal administration.

5. The use according to any one of claims 1 to 3, characterized in that: The inhibitor is in the form of a liquid dosage form, a solid dosage form, a semisolid dosage form, or a liposome.

6. The use according to any one of claims 1 to 3, characterized in that: The inhibitor is in the form of solution, emulsion, suspension, injection, eye drops, nasal drops, lotion, liniment, tablet, capsule, powder, granule, granule, pellet, drop pill, suppository, film, patch, aerosol, spray, ointment, gel, or paste.

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