CN106188244B - Short peptide therapeutic agent for inhibiting cancer cell activity and pharmaceutical composition containing the same - Google Patents
Short peptide therapeutic agent for inhibiting cancer cell activity and pharmaceutical composition containing the same Download PDFInfo
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Abstract
The invention relates to a short peptide therapeutic agent and a pharmaceutical composition containing the same, wherein the short peptide therapeutic agent comprises at least one short peptide of amino acid sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2, and any one of the short peptides comprises NO more than 40 amino acid residues, so as to inhibit activities such as proliferation of cancer cells, cancer stem protocytosis, migration, invasion, metastasis or drug resistance.
Description
Technical Field
The present invention relates to a peptide and its application, in particular, it relates to a short peptide therapeutic agent for inhibiting cancer cell activity and its medicinal composition for inhibiting cancer cell activity.
Background
Cancer patients who have metastasized or have malignant tumors that cannot be eradicated by surgery may further undergo chemotherapy after surgical removal of the primary tumor site to inhibit proliferation and metastasis of the underlying cancer cells. However, even cancer patients claiming successful chemotherapy still do not rule out the possibility of cancer recurrence or even the development of drug resistant cancer. Patients with recurrent cancer, particularly those resistant to chemotherapy, are at increased risk because the recurrent cancer cells metastasize more rapidly.
In the case of tumor (carcinoma), it is known that cancer cells stimulate the microenvironment around the tumor to produce various inflammatory factors, leukocytes, vascular hyperproliferation, proteases, etc., and the chronic inflammatory response of cancer is also related to the growth, metastasis and invasion of cancer cells.
In addition to the involvement of the tumor microenvironment in the inflammatory response, other studies have suggested that the tumor microenvironment is closely associated with tumor metastasis and chemotherapeutic resistance. The tumor microenvironment is composed of various stromal cells and other cells of different types, which can protect the tumor, and allow the tumor cells to escape and resist immune cells, resulting in drug resistance of the tumor cells.
In addition, the present inventors have studied in the past to show that when the cancer cell has a reduced CCAAT/enhancer binding protein (CCAAT/enhancer binding protein delta; CEBPD), which is a transcription factor, it is also helpful to promote further canceration, and that reactivation of CEBPD expression is shown, possibly inhibiting the growth of cancer cells.
After fibroblast and macrophage in the stroma tissue around the tumor are activated by CEBPD, it induces the production of secreted factor-pentraxin-related protein (PTX 3), which has the activity of promoting angiogenesis and can increase the migration and invasion ability of breast cancer cell, lung cancer cell and nasopharyngeal cancer cell. In addition, the present inventors have demonstrated that activation of CEBPD in cells of the tissue surrounding cancer may also promote metastasis of the cancer, even in the presence of chemotherapy to produce resistant cancer cells that grow faster and metastasize more easily.
Although some small molecule anticancer drugs, such as cis-diamminedichloroplatinum (II); CDDP; trade name Cisplatin (Cisplatin)), paclitaxel (paclitaxel; trade name Taxol) and 5-Fluorouracil (5-Fluorouracil; 5-FU), are currently available on the market, recent studies have found that these small molecule anticancer drugs not only activate CEBPD expression in cancer cells, but also activate CEBPD expression in macrophages and fibroblasts, rather, they promote drug resistance and rapid metastasis of cancer cells, resulting in poor cancer treatment effects.
In view of the above, there is a need to develop a small molecule anticancer drug to overcome the problems of the conventional small molecule anticancer drug, such as drug resistance and rapid metastasis of tumor cells.
Disclosure of Invention
One aspect of the present invention provides a short peptide therapeutic agent comprising at least one short peptide, and any one of the short peptides comprises no more than 40 amino acid residues.
Another aspect of the present invention is to provide a pharmaceutical composition comprising a short-peptide therapeutic agent and a pharmaceutically acceptable carrier, wherein the short-peptide therapeutic agent is an active ingredient and comprises at least one short peptide.
In another aspect of the present invention, a short-peptide therapeutic agent is provided for use in preparing a pharmaceutical composition for inhibiting activity of cancer cells, wherein the pharmaceutical composition comprises the short-peptide therapeutic agent and a pharmaceutically acceptable carrier, the short-peptide therapeutic agent is an active ingredient and comprises at least one short peptide for inhibiting activity of cancer cells.
According to the above aspect of the present invention, a short peptide therapeutic agent is provided. In one embodiment, the short peptide therapeutic agent can comprise at least one short peptide of the amino acid sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2, and any one of the short peptides comprises NO more than 40 amino acid residues.
According to another aspect of the present invention, a pharmaceutical composition is provided. In one embodiment, the pharmaceutical composition comprises a short-peptide therapeutic agent and a pharmaceutically acceptable carrier, wherein the short-peptide therapeutic agent is an active ingredient and comprises at least one of the amino acid sequences shown as SEQ ID NO 1 and SEQ ID NO 2, and any short peptide of the short-peptide therapeutic agent comprises NO more than 40 amino acid residues.
According to another aspect of the present invention, a method for inhibiting cancer cell activity is provided, wherein the method comprises the steps of administering a short-peptide therapeutic agent as an active ingredient to a subject in need thereof, wherein the short-peptide therapeutic agent is a pharmaceutically acceptable carrier. In one embodiment, the short-peptide therapeutic agent comprises at least one short peptide of the amino acid sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2, and any one of the short peptides comprises NO more than 40 amino acid residues, so as to specifically inhibit the activity of cancer cells in vitro.
According to an embodiment of the present invention, the pharmaceutical composition can be administered by subcutaneous injection, intratumoral injection, intravenous injection or oral route.
According to an embodiment of the present invention, the cancer cells may include, but are not limited to, breast cancer, lung cancer, nasopharyngeal cancer, epithelial cancer, and any combination thereof.
According to one embodiment of the present invention, the activities include proliferation, cancer stem cell (cancer stem), migration, invasion, metastasis or drug resistance.
The short peptide therapeutic agent for inhibiting cancer cells and the pharmaceutical composition containing the same comprise at least one short peptide, wherein any one of the short peptides comprises no more than 40 amino acid residues, so as to inhibit the activity of PTX3, and further specifically inhibit the activities such as proliferation, cancer stem protocytosis, migration, invasion, metastasis or drug resistance of cancer cells.
Drawings
In order to make the aforementioned and other objects, features, advantages and embodiments of the invention more comprehensible, thereof, the accompanying drawings are provided, and the detailed description thereof is as follows:
FIG. 1A is a schematic representation of the amino acid sequence design of a short peptide according to several embodiments of the present invention.
FIG. 1B is a bar graph showing the cell survival (%) after co-culturing in vitro several short peptides of the first embodiment of the present invention with breast cancer cells MB231 for 24 hours (top panel) or 48 hours (bottom panel).
Fig. 1C shows images of cell pellets formed after culturing breast cancer cells MB231 for 2 weeks in vitro using the short peptides of the first embodiment of the present invention (left panel) and a histogram of cell pellets (right panel) (magnification 100-fold).
FIG. 1D is a bar graph showing the survival (%) of breast cancer cells MB231 of example two after being cultured with short peptides of example one for one week, which are resistant to CDDP (left panel) or 5-FU (right panel).
FIG. 1E shows a histogram of transitional cells (left) or invasive cells (right) of the drug-resistant breast cancer cells MB231R (MBR) of the second embodiment after culturing with short peptides of the first embodiment of the invention.
Fig. 2A shows an image (left) of cell pellets formed after lung cancer cells a549 were cultured in vitro for 2 weeks using several short peptides according to the first embodiment of the present invention and a histogram (right) of cell pellets (magnification: 100 times).
FIG. 2B is a bar graph showing the cell survival (%) of lung cancer cell A549 of example two after one week of incubation with short peptides of example one of the present invention, resistant to CDDP (left panel) or 5-FU (right panel).
FIG. 2C is a bar graph showing the number of transitional cells (left) or invasive cells (right) of lung cancer cell A549 obtained in example two after culturing with short peptides of example one.
FIG. 3A shows an image (left) of cell pellets formed after 2 weeks of in vitro culture of nasopharyngeal carcinoma cell HONE1 using short peptides according to example one of the present invention and a histogram (right) of cell pellets (magnification 100 times).
FIG. 3B is a bar graph showing the cell survival (%) of the nasopharyngeal carcinoma HONE1 cells of example two after one week of incubation with short peptides of example one of the present invention, resistant to CDDP (left panel) or 5-FU (right panel).
FIG. 3C is a bar graph showing the number of transitional cells (left) or invasive cells (right) of the nasopharyngeal carcinoma HONE1 cells of example two after culturing with short peptides of example one.
FIG. 4A is a graph showing the reduction of tumor size in vivo by using short peptides of the first embodiment of the invention (left panel) or CDDP (right panel) in combination with drug-resistant breast cancer cell 4T 1R.
FIG. 4B shows a bar graph of the number of metastatic nodules per lung (left panel) and tumor weight (right panel) in vivo for FIG. 4A.
FIG. 4C shows the tumor images of the different organs in vivo of FIG. 4A and their epifluorescence radiation efficiencies.
FIG. 4D is a graph showing the reduction of tumor size in vivo by using short peptides of the first embodiment of the invention (left panel) or CDDP (right panel) in combination with drug-resistant breast cancer cells MB231R (MBR).
FIG. 4E depicts a bar graph of the number of metastatic nodules per lung (left panel) and tumor weight (right panel) in vivo for FIG. 4D.
FIG. 4F shows the tumor images of the different organs in vivo of FIG. 4D and their epifluorescence radiation efficiencies.
FIG. 5A depicts amino acid sequence design of short peptides according to several embodiments.
FIG. 5B is a bar graph showing the number of migrated cells (left) or invaded cells (right) of the euPTX 3-induced mastocarcinoma cells MB231 induced in vitro using the short peptides and polypeptides of several embodiments of the present invention.
Detailed Description
As described above, the present invention provides a short peptide therapeutic agent comprising at least one short peptide, any one of which comprises not more than 40 amino acid residues, to specifically inhibit the activity of pentraxin-related protein (PTX 3).
As used herein, the term "short peptide" refers to a short polypeptide chain having a total number of amino acid residues of no more than 40. In one embodiment, the short peptide therapeutic agent can comprise at least one short peptide of the amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2, wherein the amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 are referred to the sequence of Genbank accession No. NP-002843.2, which is incorporated herein by reference. In another embodiment, the short peptide therapeutic agent can be used in combination with two short peptides having amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2. It is stated that if the total number of amino acid residues in a polypeptide exceeds 40 (e.g., a polypeptide comprising both SEQ ID NO:1 and SEQ ID NO: 2), the resulting polypeptide is difficult to increase in effective dosage due to its large molecular weight and also has ineffective peptide fragments, which prevents subsequent application to short peptide therapeutics.
In one embodiment, the short peptides of the amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 can be produced by any known method, such as artificially synthesizing the peptides, or by using recombinant proteins obtained by expressing recombinant genes in an expression system. The methods for producing synthetic peptides or recombinant proteins are well known to those skilled in the art, and will not be described herein.
The term "specifically inhibits the binding of PTX3 receptor to PTX 3" as used herein refers to the means by which a short-peptide therapeutic agent specifically inhibits the binding of PTX3 receptor to endogenous PTX3 by means of either depletion or competitive inhibition.
In one example, the PTX3 is a soluble receptor and the short-peptide therapeutic agent can inhibit the activity of cancer cells by reducing the chance that endogenous (or full-length) PTX3 is activated or competes with endogenous PTX3 for binding to its receptor by "competitive inhibition" of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2.
In yet another example, the short peptide therapeutic agent can be used in combination with two short peptides of the amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 to further inhibit the activity of cancer cells. Here, it is stated that the short peptides of the amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 must be non-glycated. If the short peptide is pre-glycated, which itself corresponds to a biological activity comparable or comparable to endogenous PTX3, the activity of endogenous PTX3 cannot be inhibited by consumption inhibition or competitive inhibition.
The "cancer cell" referred to herein may include, but is not limited to, breast cancer, lung cancer, nasopharyngeal cancer, epithelial cancer, or any combination thereof. The term "activity" of cancer cells as used herein means that the cancer cells treated with the short-peptide therapeutic agent of the present invention can significantly inhibit the proliferation of cancer cells, the stem cell property of cancer cells, the migration of cancer cells, the invasion of cancer cells, metastasis, drug resistance, and the like.
The invention also provides a pharmaceutical composition comprising a short-peptide therapeutic agent and a pharmaceutically acceptable carrier, wherein the short-peptide therapeutic agent is as described above as an active ingredient.
In use, the short-peptide therapeutics of the invention are administered in an effective amount in combination with a pharmaceutically acceptable carrier. As used herein, an "effective dose" refers to a dose of, for example, 5mg/mL to 20mg/mL administered three times a week. In another embodiment, the effective amount is 5mg/mL to 15 mg/mL. It is stated that an effective dose of the short peptide below 5mg/mL is not effective to reduce or inhibit the binding of PTX3 receptor to PTX3 within a predetermined time.
As used herein, "pharmaceutically acceptable carrier" refers to a carrier, diluent, adjuvant and/or vehicle that is not an active ingredient per se, but is a carrier, diluent, adjuvant and/or vehicle used to deliver an active ingredient to a subject, or is added to such a composition to improve handling or storage properties of the composition, or to allow or facilitate the formation of dosage units of the composition into an excipient or any substance suitable for pharmaceutical compositions and convenient for administration. The aforementioned pharmaceutically acceptable carriers should not destroy the pharmacological activity of the active ingredient and should be non-toxic when delivering sufficient therapeutic doses of the active ingredient.
The aforementioned suitable pharmaceutically acceptable carriers can be well known to those of ordinary skill in the art of making pharmaceutical compositions and include, but are not limited to, buffers, diluents, disintegrants, binders, adhesives, humectants, polymers, lubricants, glidants, substances added to mask or counteract an unpleasant taste or odor, dyes, fragrances, and substances added to improve the appearance of the composition. Examples of the aforementioned pharmaceutically acceptable carriers can include, but are not limited to, citrate buffers, phosphate buffers, acetate buffers, bicarbonate buffers, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, magnesium carbonate, talc, gelatin, acacia, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulosic materials (such as cellulose esters and alkyl esters of alkanoic acids), low melting waxes, cocoa butter, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), ethylenediaminetetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), sodium chloride or other salts, liposomes, glycerin or powders, polymers (such as polyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol), and other pharmaceutically acceptable materials.
In use, the above-mentioned short peptide therapeutic agent and a pharmaceutical composition containing the same can be administered by subcutaneous injection, intratumoral injection, intravenous injection or oral route, thereby specifically inhibiting PTX3 activity to inhibit the activity of cancer cells. Specifically, it was confirmed by in vitro cell experiments and in vivo animal experiments that the short peptide therapeutic agent of the present invention and the pharmaceutical composition containing the same can inhibit the activity of cancer cells, such as the proliferation, the tumorigenicity, the migration, the invasion or the drug resistance of cancer cells, after being used for a predetermined period of time, such as 4 to 10 weeks.
Since the short-peptide therapeutic agent of the present invention comprises at least one short peptide, and any one of the short peptides comprises not more than 40 amino acid residues, it can specifically inhibit activities such as proliferation of cancer cells, cancer stem cell activity, migration, invasion, metastasis or drug resistance as well as the activity of PTX3, and thus the short-peptide therapeutic agent can be applied to the preparation of a pharmaceutical composition for specifically inhibiting PTX3 activity.
The following examples are provided to illustrate the present invention, but not to limit the invention, and those skilled in the art can make various modifications and changes without departing from the spirit and scope of the invention.
The first embodiment is as follows: preparation of short peptide therapeutic agent
In this example, artificially synthesized short peptide RI37 (having the amino acid sequence shown in SEQ ID NO: 1), short peptide GI40 (having the amino acid sequence shown in SEQ ID NO: 2), and short peptide KT44 (having the amino acid sequence shown in SEQ ID NO: 3) were used as short peptide therapeutics, respectively, and polypeptide pPTX3/C (having the amino acid sequence shown in SEQ ID NO: 4) was used as a control group. The sequence design of short peptide RI37, short peptide GI40, short peptide KT44 and polypeptide euPTX3 is shown in FIG. 1A, and the sequence design of short peptide RI37, short peptide GI40, polypeptide pPTX3/C and polypeptide euPTX3 is shown in FIG. 5A.
The short peptide RI37, the short peptide GI40, and the short peptide KT44 can be synthesized by, for example, Koroth International science and technology Co., Ltd. The above pPTX3/C is a recombinant protein which is expressed and purified by the present inventors by themselves using a known prokaryotic expression system (E.coli). The above polypeptide euPTX3 can be purified, for example, from mouse myeloma cells, PTX3, which is commercially available (R & D system Inc.).
Example two: establishing a cellular assay model
This example utilizes a human breast cancer cell line (MDA-MB231, deposited at the center for biological resource conservation and research (BCRC) of New bamboo food industry development institute of Taiwan, FIRDI, food agency No. 331 of 300 New bamboo City, China, with the deposit number BCRC 60425, also deposited at the American type culture Collection (ATCC, P.O. Box1549, Manassas, VA 20108, USA), with the deposit number ATCC HTB-26, hereinafter referred to as MB231) or a cisplatin-resistant breast cancer cell line (CDDP-resistant MDA-MB231, MDA-MB231 MBR 231R, hereinafter referred to as MBR), a human lung cancer cell line A549 (deposit number BCRC 60074, ATCC CCL-185), human nasopharyngeal carcinoma cell line HONE1(int.J.cancer.1990 Jan 15; 45(1): 83-9; Proc.Natl.Acad.Sci.USA, Vol.86, pp.9524-9528, December 1989) and the like, and the inhibitory effect of the short-peptide therapeutic agent of example one on cancer cell activity was evaluated.
For example, the MDA-MB231 cells, MBR cells, 4T1 cells, 4T1R cells and A549 cells are cultured in Dulbecco's modified Eagle medium (DMEM; Gibco Co.) cell culture solution containing 10% Fetal bovine serum albumin (FBS) and antibiotics (50-100. mu.g/mL streptomycin and 50-100U/mL penicillin), and the HONE1 cells are cultured in RPMI-1640 cell culture solution containing 10% FBS and antibiotics and are cultured at 37 ℃ under 5% carbon dioxide, which is known by any person of ordinary skill in the art and will not be described herein.
Example three: inhibition of cancer cell activity by short-peptide therapeutics
The short-peptide therapeutic agent of example one was added to the cells of example two, respectively, and then co-cultured, and then the evaluation of the correlation was performed in the following manner.
1. Evaluation of the Effect of short peptides on cancer cell survival
After culturing the short peptide RI37, short peptide GI40 and short peptide KT44 of example I and the breast cancer cell MB231 of example II for 24 hours or 48 hours, cytotoxicity was tested using MTT [3- (4, 5-dimethylthiozol-2-yl) -2, 5-diphenyltetrazolium boss ] (Sigma). MTT is tetrazolium salt (tetrazolium salt), and blue formazan (formazan) crystals are generated from transparent and colorless material after mitochondrion decomposition in living cells, and the effect of the short peptide of example one on the proliferation of the breast cancer cell MB231 of example two was determined by setting the cell survival rate without the short peptide of example one as 100%.
Referring to FIG. 1B, FIG. 1B is a bar graph showing the cell survival (%) after culturing several short peptides of the first embodiment of the present invention in vitro with breast cancer cells MB231 for 24 hours (top panel of FIG. 1B) or 48 hours (bottom panel of FIG. 1B), wherein the graph number "below the horizontal axis represents that no specific short peptide is added during the cell culture, and the triangles represent that the amount of the specific short peptide is increased. As can be seen from the results in fig. 1B, the short peptide RI37, the short peptide GI40, and the short peptide KT44 according to the first embodiment of the present invention have no significant effect on the cell survival rate after being cultured with the breast cancer cell MB231 for 24 hours or 48 hours, respectively.
2. Assessing the effect of short peptides on the oncogenic properties of cancer cells
The breast cancer cell MB231, the lung cancer cell A549 and the nasopharyngeal carcinoma cell HONE1 have cancer stem protocytogenic property, and the cell pellet (sphere) of the cancer cell can be formed by adding the polypeptide euPTX 3. The effect of the short peptides of example one on the cancer stem protocytogenicity of cancer cells was evaluated by co-culturing the short peptides RI37, GI40, KT44 and euPTX3 of example one with the cancer cells.
The short peptide RI37, short peptide GI40, short peptide KT44 (all short peptides are 10. mu.g/mL, 225nM) and polypeptide euPTX3 (2.5. mu.g/mL) of the first example were mixed with the cell density of 5X 103The breast cancer cells MB231, lung cancer cells A549 and nasopharyngeal cancer cells HONE1 were cultured in a cell culture medium DMEM/F12(Gibco) without serum (serum-free) [ containing B27(Invitrogen), epidermal Growth Factor (EGF; Abcam) at 20 ng/mL, and basic Fibroblast Growth Factor (basic Fibroblast Growth Factor, bFGF; Peprotech) at 10ng/mL in a multi-well plate (Multi-well plate with ultra-low-surface-adherence surface Multi surface culture; Corning Inc.)]Then, co-cultivation is performed. After 2 weeks of culture, the number of cell pellets was observed by an optical microscope.
Refer to fig. 1C, 2A and 3A. FIG. 1C shows the image (left) and histogram (right) of the formation of cell pellets after 2 weeks of in vitro culture of breast cancer cells MB231 with short peptides according to the first embodiment of the present invention. Fig. 2A shows images of cell pellets formed after lung cancer cells a549 were cultured in vitro using several short peptides of the first example of the invention for 2 weeks (left panel) and cell pellet number histogram (right panel). FIG. 3A shows the image (left) and histogram (right) of cell pellets formed after 2 weeks of in vitro culture of nasopharyngeal carcinoma cell HONE1 using short peptides of the first embodiment of the present invention. FIGS. 1C, 2A and 3A are all at 100-fold magnification, and the graph number "below the horizontal axis represents that no specific short peptide was added during cell culture.
As can be seen from the results of fig. 1C, fig. 2A and fig. 3A, compared to the group to which only the polypeptide euPTX3 is added, the group to which the short peptide RI37 and the short peptide GI40 are added does indeed inhibit the number of cell pellets caused by the polypeptide euPTX3 after the short peptide RI37, the short peptide GI40 and the polypeptide euPTX3 are cultured together with the breast cancer cell MB231, the lung cancer cell a549 and the nasopharyngeal cancer cell HONE1, and the difference is statistically significant. However, the addition of the short peptide KT44 cannot inhibit the number of cell pellets caused by the polypeptide euPTX 3.
3. Assessing the effect of short peptides on drug resistance of cancer cells
The breast cancer cell MB231, the lung cancer cell A549 and the nasopharyngeal carcinoma cell HONE1 have drug resistance, and the addition of the polypeptide euPTX3 can promote the drug resistance of the cancer cells. The effect of the short peptides of example one on the resistance of cancer cells was evaluated by co-culturing the short peptides RI37, GI40, KT44 and euPTX3 of example one with the cancer cells.
The short peptide RI37, short peptide GI40 and short peptide KT44 (all 10 μ g/mL, 225nM) of example I were mixed with 1X 104The breast cancer cell MB231, the lung cancer cell A549 and the nasopharyngeal carcinoma cell HONE1 with a plurality of cell numbers are cultured together. After 1 week of culture, 40. mu.M of Cisplatin (CDDP) or 100. mu.M of 5-fluorouracil (5-FU) was added thereto, respectively, followed by culture for another 48 hours. The influence of the short peptide of example one on the drug resistance of the cancer cells of example two was evaluated by the aforementioned MTT assay, with the cell viability of the polypeptide euPTX3 (2.5. mu.g/mL) of example one added alone as 100%.
Refer to fig. 1D, 2B and 3B. FIG. 1D is a bar graph showing the survival (%) of breast cancer cells MB231 of example two after being cultured with short peptides of example one for one week, which are resistant to CDDP (left panel) or 5-FU (right panel). FIG. 2B is a bar graph showing the cell survival (%) of lung cancer cells A549 of example two after one week of culture using the short peptides of example one of the present invention, resistant to CDDP (left panel) or 5-FU (right panel). FIG. 3B is a bar graph of cell survival (%) after one week of nasopharyngeal carcinoma HONE1 cells of example two cultured with short peptides of example one of the present invention, resistant to CDDP (left panel) or 5-FU (right panel). The numbers "below the horizontal axes in FIGS. 1D, 2B and 3B represent that no specific short peptide was added during cell culture.
From the results shown in FIG. 1D, FIG. 2B and FIG. 3B, it is clear that the group to which only the polypeptide euPTX3 was added increased the resistance of the breast cancer cell MB231, the lung cancer cell A549 and the nasopharyngeal cancer cell HONE1 to CDDP or 5-FU and the cancer cell survival (%) as compared with the group to which the polypeptide euPTX3 was not added.
However, the group of addition of short peptide RI37 and short peptide GI40 did inhibit drug resistance caused by polypeptide euPTX3, and the difference was statistically significant, wherein the effect of short peptide RI37 inhibition was better, and the effect of short peptide GI40 was less, while addition of short peptide KT44 did not inhibit drug resistance caused by polypeptide euPTX 3.
4. Evaluation of the Effect of short peptides on cancer cell migration
The migration of the breast cancer cell MB231, the lung cancer cell A549 and the nasopharyngeal carcinoma cell HONE1 can be promoted (migration), and the migration of the cancer cell can be promoted by adding the polypeptide euPTX 3. The effect of the short peptides of example one on the migration of cancer cells was evaluated by co-culturing the short peptides RI37, GI40, KT44, euPTX3 and pPTX3/C of example one with the cancer cells.
The short peptide RI37, short peptide GI40 and short peptide KT44 of example I were combined with 5X 10 of the monoclonal antibody against the breast cancer cell MB231, lung cancer cell A549 and nasopharyngeal cancer cell HONE1, respectively4The effect of several short peptides of example one on the migration of cancer cells of example two was judged by seeding the upper layer (containing microwells with a pore size of 8 μm) of a 24-well cell culture dish for 19 hours at a cell/well cell density.
Will be 5X 104Cell/well cell density of breast cancer cell MB231, lung cancer cell A549 or nasopharyngeal carcinoma cell HONE1 at 5 × 104Cell density of cells/well was seeded on the upper layer of a Bowden cell-walker (Boyden chamber) and cultured for 3 hours.
Next, the upper cell culture medium was replaced with a serum-free cell culture medium, and the short peptide RI37, the short peptide GI40, the short peptide KT44, the polypeptide euPTX3, and the polypeptide pPTX3/C of example one were added to the lower serum-free cell culture medium. After 16 hours of culture, cells on the inner side of the upper layer were scraped with a cotton swab, stained with 4 ', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI; Invitrogen), and the number of cells moving to the outer side of the bottom of the upper layer was counted under a field of view at a magnification of 200 times of a fluorescence microscope.
Refer to fig. 1E, 2C, 3C and 5B. FIG. 1E is a bar graph showing the number of transitional cells of the second embodiment of the drug-resistant breast cancer cell MB231R (MBR) cultured with the short peptides of the first embodiment of the invention. FIG. 2C is a bar chart showing the number of transitional cells of lung cancer cell A549 obtained in example two after culturing with short peptides of example one. FIG. 3C is a bar graph showing the number of transitional cells of nasopharyngeal carcinoma HONE1 cells of example two after culturing with short peptides of example one. Wherein, the addition amount of the polypeptide euPTX3 in the left picture of figure 1E, the left picture of figure 2C and the left picture of figure 3C is 2.5 mug/mL, and the addition amount of the short peptide RI37, the short peptide GI40 and the KT44 is 10 mug/mL.
FIG. 5B is a bar graph showing the number of migrated cells of the breast cancer cell MB231 of the second embodiment after culturing with the short peptides and polypeptides of the first embodiment of the present invention, wherein the amount of the polypeptide euPTX3 added is 2.5. mu.g/mL, the amount of the short peptide RI37 added is 3.7. mu.g/mL, the amount of the short peptide GI40 added is 4. mu.g/mL), and the amount of the polypeptide pPTX3/C added is 10. mu.g/mL, such that the number of molecules of the polypeptide euPTX3, the short peptide RI37, the short peptide GI40 and the polypeptide pPTX3/C are the same. The numbers "below the horizontal axis of the left panels in FIG. 1E, 2C, 3C and 5B represent that no specific short peptide was added during cell culture.
From the results of the left panels of fig. 1E, 2C, 3C and 5B, it is clear that the group added with the polypeptide euPTX3 alone can increase the number of migrated cells of the drug-resistant breast cancer cell MB231R, lung cancer cell a549 and nasopharyngeal cancer cell HONE1, compared to the group without the polypeptide euPTX 3. However, the group of addition of short peptide RI37 and short peptide GI40 did inhibit the number of migrated cells caused by polypeptide euPTX3, and this difference was statistically significant, but addition of short peptide KT44 did not inhibit the number of migrated cells caused by polypeptide euPTX 3.
In addition, the left panel of fig. 5B further demonstrates that the group with both short peptide RI37 and short peptide GI40 further inhibited the number of migrated cells caused by polypeptide euPTX3 compared to the group with only short peptide RI37 or short peptide GI 40. Although the addition of the polypeptide pPTX3/C can further inhibit the number of transitional cells caused by the polypeptide euPTX3, the polypeptide pPTX3/C has a larger molecule and contains fragments of the short peptide RI37, the short peptide KT44 and the short peptide GI40, which is difficult to be applied to a short peptide therapeutic agent in the future and is difficult to improve the effective dose due to the larger molecular weight. Secondly, although pPTX3/C prepared by E.coli has no introduction of activated glycosyl, considering the problem that polypeptide generated by E.coli is easy to be polluted by endotoxin (such as LPS), the polypeptide has a large risk when being directly used for treatment, so that the short peptide RI37, the short peptide KT44 and the short peptide GI40 are directly chemically synthesized in an artificial mode, and the advantages of the polypeptide are that the molecular weight is small, and the problem of endotoxin pollution is also solved.
5. Evaluation of the Effect of short peptides on the invasive ability of cancer cells
The breast cancer cell MB231, the lung cancer cell A549 and the nasopharyngeal carcinoma cell HONE1 can invade, and the invasion of the cancer cells can be promoted by adding the polypeptide euPTX 3. The effect of the short peptides of example one on the invasion of cancer cells was evaluated by co-culturing the short peptides RI37, GI40, KT44, euPTX3 and pPTX3/C of example one with the cancer cells.
Will be 5X 104Cell/well cell density of breast cancer cell MB231, lung cancer cell A549 or nasopharyngeal carcinoma cell HONE1 at 5 × 104Cell density of cells/well seeded on the upper layer of a Bowden cell-transplanter (Boyden chamber) with a pre-coated basement membrane matrix (matrigel, available from matrigel) between the upper and lower layersBD Bioscience) were separated and cultured for 3 hours.
Next, the upper cell culture medium was replaced with a serum-free cell culture medium, and the short peptide RI37, the short peptide GI40, the short peptide KT44, the polypeptide euPTX3, and the polypeptide pPTX3/C of example one were added to the lower serum-free cell culture medium. After 16 hours of culture, cells on the inner side of the upper layer were scraped with a cotton swab, stained with 4', 6-diamidino-2-phenylindole (DAPI; Invitrogen), and the number of cells moving to the outer side of the bottom of the upper layer was counted under a field of view at a magnification of 200 times of a fluorescence microscope.
Refer to fig. 1E, 2C, 3C and 5B. FIG. 1E shows on the right a histogram of the number of invasive cells of the drug-resistant breast cancer cells MB231R (MBR) of the second embodiment cultured with the short peptides of the first embodiment of the present invention. FIG. 2C is a bar chart showing the number of invaded cells of lung cancer cell A549 obtained in example two after culturing with short peptides of example one. FIG. 3C is a bar graph showing the number of invasive cells of HONE1 of nasopharyngeal carcinoma of example two after culturing with short peptides of example one. Wherein, the addition amount of the polypeptide euPTX3 in the right picture of figure 1E, the right picture of figure 2C and the right picture of figure 3C is 2.5 mug/mL, and the addition amount of the short peptide RI37, the short peptide GI40 and the KT44 is 10 mug/mL.
FIG. 5B is a bar graph showing the number of invasive cells of the breast cancer cell MB231 of the second embodiment after culturing with the plurality of short peptides and polypeptides of the first embodiment of the invention, wherein the amount of the polypeptide euPTX3 added in the right graph of FIG. 5B is 2.5. mu.g/mL, the amount of the short peptide RI37 added is 3.7. mu.g/mL, the amount of the short peptide GI40 added is 4. mu.g/mL), and the amount of the polypeptide pPTX3/C added is 10. mu.g/mL, such that the number of the molecules of the polypeptide euPTX3, the short peptide RI37, the short peptide GI40 and the polypeptide pPTX3/C are the same. The numbers "below the horizontal axis of the right panels of FIG. 1E, 2C, 3C and 5B represent that no specific short peptide was added during cell culture.
From the results of the right panel of fig. 1E, the right panel of fig. 2C, the right panel of fig. 3C, and the right panel of fig. 5B, it is clear that the group to which the polypeptide euPTX3 was added increases the number of invasive cells of the drug-resistant breast cancer cell MB231R (MBR), the lung cancer cell a549, and the nasopharyngeal cancer cell HONE1, compared to the group to which the polypeptide euPTX3 was not added. However, the group added with short peptide RI37 and short peptide GI40 did inhibit the number of cells invaded by polypeptide euPTX3, and the difference was statistically significant, but the group added with short peptide KT44 did not inhibit the number of cells invaded by polypeptide euPTX 3.
In addition, fig. 5B further demonstrates that the group added with short peptide RI37 and short peptide GI40 further inhibited the number of cells affected by polypeptide euPTX3 compared to the group added with short peptide RI37 or short peptide GI40 alone. Although the addition of the polypeptide pPTX3/C can further inhibit the cell invasion number caused by the polypeptide euPTX3, the polypeptide pPTX3/C has larger molecules and contains fragments of short peptide RI37, short peptide KT44 and short peptide GI40, which is difficult to be applied to short peptide therapeutics in the follow-up process, and the effective dose is difficult to increase due to the larger molecular weight. Secondly, although pPTX3/C prepared by E.coli has no introduction of activated glycosyl, considering the problem that polypeptide generated by E.coli is easy to be polluted by endotoxin (such as LPS), the polypeptide has a large risk when being directly used for treatment, so that the short peptide RI37, the short peptide KT44 and the short peptide GI40 are directly chemically synthesized in an artificial mode, and the advantages of the polypeptide are that the molecular weight is small, and the problem of endotoxin pollution is also solved.
Example four: evaluation of the Effect of short peptides on tumors Using animal test model
1. Establishing animal test mode
This example is to mix 1X 106The mCherry red fluorescent protein labeled mouse drug-resistant breast cancer cell strain [ mCherry fluorescent CDDP-resistant mouse 4T1(ATCC CRL-2539), 4T1R]The dorsal posterior side of BALB/c female mice (purchased from the animal center of Taiwan university of success) 6 to 8 weeks old were inoculated subcutaneously. After 1 week of inoculation, mice were intraperitoneally injected 1 time a week with 5mg/kg CDDP (dissolved in 1% (w/v) DMSO) or only 1% (w/v) DMSO and either not intratumorally injected or 3 times a week with the short peptide RI37, KT44 and GI40 of example one (all 50 μ g), sacrificed 6 weeks after inoculation of cancer cells, the spleen, kidney, lung and liver were removed, their tumor weights were measured (6 replicates per group), and the IVIS spectral Imaging System (IVIS spectral Imaging System 200, Caliper) was used) The metastatic activity of cancer cells and the number of metastatic nodules of each organ were determined (number of metastatic nodules per organ). Tumor size was measured by a commercially available external caliper, and tumor volume (V) was calculated using the following formula (I):
V＝(w×l2)×0.52 (I)
in formula (I), w is the tumor width and l is the tumor length. Each experimental condition was repeated at least three times.
In addition, 1 × 106The mCherry red fluorescent protein-labeled human drug-resistant breast cancer cell line (mCherry fluorescent CDDP-resistant MDA-MB-231, MB231R) of (Hakka sp.) was subcutaneously inoculated to the back side of the 6 to 8-week-old NOD-SCID female immunodeficient mice (purchased from Taiwan university of success animal center). After 1 week of inoculation, mice were intraperitoneally injected 1 time a week with 5mg/kg of CDDP (dissolved in 1% (w/v) DMSO) or only 1% (w/v) DMSO, and were not injected intratumorally or injected 3 times a week with the short peptide RI37, KT44 and GI40 of example one (all 50 μ g), mice were sacrificed 10 weeks after inoculation with cancer cells, the spleen, kidney, lung and liver were removed, the tumor weight thereof was measured (3 replicates per group), and the metastatic activity of cancer cells and the number of metastatic nodules of each organ were judged using the IVIS spectral imaging system. Tumor size was measured by a commercially available external caliper and tumor volume (V) was calculated using formula (I) above. Each experimental condition was repeated at least three times.
2. Evaluation of the Effect of short peptides on allografted tumors
Referring to FIG. 4A, a graph of the reduction of size of 4T 1R-resistant breast cancer tumors in vivo using a combination of short peptides of embodiment one of the present invention (left panel) and CDDP (right panel) is shown. From the results shown in fig. 4A, it can be seen that both short peptide RI37 and short peptide GI40 of the first embodiment of the present invention can inhibit tumor growth and reduce drug resistance of tumors caused by drug-resistant breast cancer cells 4T1R, wherein the inhibition effect of short peptide RI37 is better, short peptide GI40 times is less, and short peptide KT44 has no significant inhibition effect.
Referring to FIG. 4B, a bar graph of the number of metastatic nodules per lung (left panel) and tumor weight (right panel) in vivo for FIG. 4A is shown. From the results shown in fig. 4B, it can be seen that both short peptide RI37 and short peptide GI40 of the first embodiment of the present invention can inhibit tumor metastasis and growth, wherein the effect of short peptide RI37 is better, the effect of short peptide GI40 times is less, and the effect of short peptide KT44 is not significantly inhibited.
Referring to fig. 4C, an image of a tumor of the different organs in vivo of fig. 4A and its epifluorescence (epifluorescence) radiation efficiency are shown. As can be seen from the results in fig. 4C, the short peptide RI37 and the short peptide GI40 of the first embodiment of the present invention both inhibited metastasis and growth of tumors in vivo, whereas the short peptide KT44 did not have significant inhibitory effect, and nodules of cancer cell metastasis were found in the lung.
3. Evaluation of the Effect of short peptides on xenograft tumors
Referring now to FIG. 4D, a graph of the reduction of MB231R (MBR) resistant breast cancer tumor size in vivo using short peptides (left panel) or CDDP in combination according to one embodiment of the present invention is shown. From the results shown in fig. 4D, it can be seen that both short peptide RI37 and short peptide GI40 of the first embodiment of the present invention can inhibit tumor growth and reduce the drug resistance of tumor caused by drug-resistant breast cancer cell MB231R (MBR), wherein the inhibitory effect of short peptide RI37 is better, the inhibitory effect of short peptide GI40 times is better, and the inhibitory effect of short peptide KT44 is not significant.
Referring to FIG. 4E, a bar graph of the number of metastatic nodules per lung (left panel) and tumor weight (right panel) in vivo for FIG. 4D is shown. From the results shown in fig. 4E, it can be seen that both short peptide RI37 and short peptide GI40 of the first embodiment of the present invention can inhibit tumor metastasis and growth, wherein the effect of short peptide RI37 is better, the effect of short peptide GI40 times is less, and the effect of short peptide KT44 is not significantly inhibited.
Referring to FIG. 4F, tumor images of different organs in vivo and their epifluorescence radiation efficiencies of FIG. 4D are shown. As can be seen from the results shown in fig. 4F, the short peptide RI37 and the short peptide GI40 of the first embodiment of the present invention both inhibited metastasis and growth of tumors in vivo, whereas the short peptide KT44 did not have significant inhibitory effect, and nodules of cancer cell metastasis were found in the lung.
The data from the above examples were obtained as the mean standard deviation of the positive and negative values of the triplicate experiments for each time point and each sample, all values were analyzed by one-way ANOVA. The data of the above examples are represented by the graph having statistical significance (p < 0.05), the graph having statistical significance (p < 0.01), and the graph having statistical significance (p < 0.001).
In summary, it is demonstrated from the above examples that the use of short peptide RI37 and/or short peptide GI40 with no more than 40 amino acid residues according to the present invention can effectively inhibit the activity of endogenous PTX3, and further specifically inhibit the activities such as proliferation of cancer cells, cancer stem cell activity, migration, invasion, metastasis or drug resistance. Therefore, the short peptide of the first embodiment of the invention can be used as a short peptide therapeutic agent to prepare a pharmaceutical composition for specifically inhibiting the activity of PTX 3.
It should be noted that although the short-peptide therapeutic agent of the present invention for inhibiting the activity of an pentraxin-related protein and the pharmaceutical composition containing the same are exemplified by short peptides in a specific range, a specific process, a specific analytical method or a specific instrument, it is understood by those skilled in the art that the present invention is not limited thereto, and the short-peptide therapeutic agent and the pharmaceutical composition containing the same can be carried out using shorter short peptides, other processes, other analytical methods or other instruments without departing from the spirit and scope of the present invention.
As is apparent from the above examples, the present invention provides a short-peptide therapeutic agent and a pharmaceutical composition containing the same, which are advantageous in that the use of short peptide RI37 and/or short peptide GI40 having not more than 40 amino acid residues can effectively inhibit the activity of endogenous PTX3, and further specifically inhibit the activities such as proliferation of cancer cells, cancer stem cell activity, migration, invasion, metastasis, or drug resistance.
Although the present invention has been described with respect to the above embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. A short peptide therapeutic agent comprises at least one short peptide consisting of the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and any one of the short peptides comprises NO more than 40 amino acid residues.
2. A pharmaceutical composition comprises a short peptide therapeutic agent and a pharmaceutically acceptable carrier, wherein the short peptide therapeutic agent is an active ingredient, the short peptide therapeutic agent is at least one short peptide consisting of an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2, and any one of the short peptides comprises NO more than 40 amino acid residues.
3. The application of a short peptide therapeutic agent in preparing a pharmaceutical composition for inhibiting the activity of cancer cells comprises the short peptide therapeutic agent and a pharmaceutically acceptable carrier, wherein the short peptide therapeutic agent is used as an active ingredient, the short peptide therapeutic agent is at least one short peptide consisting of an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2, and any one of the short peptides comprises NO more than 40 amino acid residues so as to specifically inhibit the activity of the cancer cells in vitro.
4. Use of a short peptide therapeutic according to claim 3 for the preparation of a pharmaceutical composition for inhibiting the activity of cancer cells, wherein the pharmaceutical composition is administered via subcutaneous injection, intratumoral injection, intravenous injection or oral route.
5. The use of a short peptide therapeutic of claim 3 for the preparation of a pharmaceutical composition for inhibiting the activity of a cancer cell selected from the group consisting of a breast cancer cell, a lung cancer cell, a nasopharyngeal cancer cell, and any combination thereof.
6. Use of a short peptide therapeutic according to claim 3 for the preparation of a pharmaceutical composition for inhibiting the activity of cancer cells, wherein the activity comprises cancer stemogenesis, metastasis, invasion
7. The application of a short peptide therapeutic agent in preparing a pharmaceutical composition for inhibiting the activity of cancer cells is characterized in that the pharmaceutical composition comprises the short peptide therapeutic agent and a pharmaceutically acceptable carrier, the short peptide therapeutic agent is an active ingredient, the short peptide therapeutic agent is at least one short peptide consisting of amino acid sequences shown in SEQ ID NO 1 or SEQ ID NO 2, and any one of the short peptides comprises NO more than 40 amino acid residues so as to specifically inhibit the metastasis and the growth of tumors.
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| Anti-angiogenic peptides for cancer therapeutics;Elena V.等;《Curr Pharm Biotechnol.》;20110801;第12卷(第8期);第1101–1116页 * |
| Anti-FGF2 approaches as a strategy to compensate resistance to anti-VEGF therapy: long-pentraxin 3 as a novel antiangiogenic FGF2-antagonist;Alessi P等;《Eur Cytokine Netw》;20091231;第20卷(第4期);第225–234页 * |
| Fibroblast Growth Factor 2-antagonist Activity of a Long-Pentraxin 3-derived Antiangiogenic Pentapeptide;Leali D等;《J Cell Mol Med》;20100831;第14卷(第8期);第2109-2121页 * |
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