JP2004175689A - Agent for inhibiting tumor metastasis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new agent for inhibiting tumor metastasis based on the finding that an ADAMTS-1 protein (a disintegrin and metalloproteinase with thrombospondin type 1 motiefs protein) is useful as an antimetastatic agent of cancer. <P>SOLUTION: This agent for inhibiting the tumor metastasis contains the ADAMTS-1 protein or a functionally equivalent modified body thereof, a polynucleotide encoding the ADAMTS-1 protein or the functionally equivalent modified body thereof, or a recombinant vector containing the polynucleotide as an active ingredient. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cancer metastasis inhibitor.
[0002]
[Prior art]
ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) protein has ADAM-type metalloproteinase (MMP) activity and thrombospondin motif (thrombospoin) ["The Journal of Biological Chemistry", (USA), 1997, Vol. 272, p. 556-562 (Non-Patent Document 1)]. ADAMTS-1 protein is an extracellular matrix-bound MMP, and ADAMTS-1 protein appears to be involved in the metabolism of extracellular matrix components.
[0003]
In fact, recent results of our study revealed that ADAMTS-1 protein has an activity that degrades aggrecan, which is the main component of proteoglycans in joints ["FEBS letters" ) ", (UK), 2000, 478, p. 241-245 (Non-Patent Document 2)]. In addition, it is known that MMP is involved in invasion of normal tissues in cancer metastasis ["Science", (USA), 2002, Vol. 295, p. 2387-2392 (Non-Patent Document 3)].
[0004]
[Non-Patent Document 1]
“The Journal of Biological Chemistry” (USA), 1997, Vol. 272, p. 556-562
[Non-Patent Document 2]
"FEBS letters" (UK), 2000, 478, p. 241-245
[Non-Patent Document 3]
“Science” (USA), 2002, Vol. 295, p. 2387-2392
[0005]
[Problems to be solved by the invention]
Under such circumstances, the present inventor was examining the effect of the ADAMTS-1 protein on cancer metastasis. Contrary to expectation, the ADAMTS-1 gene was introduced in a mouse experimental lung metastasis system. It was found that metastasis of cancer cells to the lung was significantly suppressed as compared with control, and ADAMTS-1 protein was useful as an antimetastatic agent for cancer. The present invention is based on such knowledge.
Accordingly, an object of the present invention is to provide a novel cancer metastasis inhibitor.
[0006]
[Means for Solving the Problems]
The object is to use an ADAMTS-1 protein or a functional equivalent variant thereof according to the present invention, a polynucleotide encoding the ADAMTS-1 protein or a functional equivalent variant thereof, or a recombinant vector containing the polynucleotide as an active ingredient. It can be solved by containing a cancer metastasis inhibitor.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The cancer metastasis inhibitor of the present invention is an active ingredient,
(1) ADAMTS-1 protein,
(2) a functional equivalent variant of ADAMTS-1 protein,
(3) a polynucleotide encoding an ADAMTS-1 protein,
(4) a polynucleotide encoding a functional equivalent variant of ADAMTS-1 protein,
(5) A recombinant vector containing a polynucleotide encoding ADAMTS-1 protein, or (6) a recombinant vector containing a polynucleotide encoding a functional equivalent variant of ADAMTS-1 protein.
[0008]
As used herein, “ADAMTS-1 protein” refers to human ADAMTS-1 protein disclosed in JP-A-11-46781, and proteins in various animals corresponding to this human ADAMTS-1 protein, such as mouse It means ADAMTS-1 protein (J. Biol. Chem., 272, 556-562, 1997). That is, it means natural ADAMTS-1 protein of various animals including human. The human ADAMTS-1 protein consists of 727 amino acid residues, and the amino acid sequence thereof is disclosed in JP-A-11-46781.
[0009]
In the present specification, “functionally equivalent variant of ADAMTS-1 protein” means deletion or substitution of one or more (preferably one or several) amino acids in the amino acid sequence of natural ADAMTS-1 protein. And / or a protein having an added amino acid sequence and having the same activity as that of the natural ADAMTS-1 protein. Here, “the same activity as ADAMTS-1 protein” means, for example, an activity that affects the hematopoietic function, for example, an activity that decreases the number of white blood cells and platelets, and at the same time increases the number of red blood cells (JP-A-11-11 46781) or aggrecanase activity (Japanese Patent Laid-Open No. 2001-302536).
[0010]
The shape of the ADAMTS-1 protein or functionally equivalent variant thereof contained in the cancer metastasis inhibitor of the present invention is not particularly limited as long as the cancer metastasis inhibitory activity is maintained. It can be the type of ADAMTS-1 protein itself, or it can be a fusion protein of an ADAMTS-1 protein or a functional equivalent variant thereof and a fusion partner (eg, protein or peptide).
Examples of the fusion partner include a protein or peptide for purification [for example, glutathione S-transferase (GST) or a histidine hexapeptide], a protein or peptide for detection [for example, β-galactosidase α peptide (LacZ)]. Or a protein or peptide for expression (eg, a signal sequence).
[0011]
The polynucleotide encoding the ADAMTS-1 protein or a functional equivalent variant thereof that can be used as an active ingredient of the cancer metastasis inhibitor of the present invention may encode the ADAMTS-1 protein or a functional equivalent variant thereof. Although it is not particularly limited as far as possible, for example, a polynucleotide chemically synthesized based on the amino acid sequence of the ADAMTS-1 gene existing in nature, or the ADAMTS-1 protein or a functionally equivalent variant thereof, etc. Can be mentioned. The term “polynucleotide” in the present specification includes both DNA and RNA.
[0012]
In the cancer metastasis inhibitor of the present invention, when ADAMTS-1 protein or a functional equivalent variant thereof is used as an active ingredient, ADAMTS-1 protein or a functional equivalent variant thereof may be used alone or as desired. It can be administered to animals, preferably mammals (especially humans) together with conventional carriers that are pharmaceutically or veterinarily acceptable.
[0013]
There are no particular limitations on the dosage form, for example, oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills, or injections And parenteral preparations such as preparations, external preparations, ointments, suppositories, topical creams, and eye drops.
[0014]
These oral preparations include, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid ester, talc, stearic acid Excipients such as magnesium, polyethylene glycol, magnesium silicate, anhydrous silicic acid, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, preservatives, coloring It can be produced according to a conventional method using an agent, a fragrance, a corrigent, a stabilizer, a humectant, an antiseptic, an antioxidant, or the like.
[0015]
Examples of parenteral administration methods include injection (subcutaneous, intravenous, etc.) or rectal administration. Of these, the injection is most preferably used.
For example, in the preparation of injections, in addition to the ADAMTS-1 protein as an active ingredient or a functional equivalent variant thereof, for example, a water-soluble solvent such as physiological saline or Ringer's solution, a water-insoluble solvent such as vegetable oil or fatty acid ester, etc. An isotonic agent such as a neutral solvent, glucose or sodium chloride, a solubilizer, a stabilizer, a preservative, a suspending agent, or an emulsifier can be optionally used.
In addition, the cancer metastasis inhibitor of the present invention may be administered using a sustained release preparation technique using a sustained release polymer or the like. For example, the cancer metastasis inhibitor of the present invention can be incorporated into an ethylene vinyl acetate polymer pellet and the pellet can be surgically implanted into the tissue to be treated or prevented.
[0016]
The cancer metastasis inhibitor of the present invention is not limited to this, but ADAMTS-1 protein or a functional equivalent variant thereof is 0.01 to 99% by weight, preferably 0.1 to 80% by weight. It can be contained in an amount.
The dosage in the case of using the cancer metastasis inhibitor of the present invention can be appropriately determined according to, for example, the type of illness, patient age, sex, body weight, degree of symptoms, or administration method, and orally. Or it can be administered parenterally.
Furthermore, the form is not limited to pharmaceuticals, and various forms such as functional foods, health foods, or feeds can be given in the form of food and drink.
[0017]
In the cancer metastasis inhibitor of the present invention, when using a polynucleotide encoding ADAMTS-1 protein or a functional equivalent variant thereof as an active ingredient, or a recombinant vector containing the same, the polynucleotide or the recombinant vector Can be administered to animals, preferably mammals (particularly humans), alone or together with conventional carriers that can be used for gene therapy if desired.
[0018]
For example, in addition to a method of directly administering a polynucleotide encoding ADAMTS-1 protein or a functional equivalent variant thereof by injection, a method of administering a recombinant vector in which the polynucleotide is incorporated can be mentioned. Examples of vectors that can be used to prepare the recombinant vectors include vectors that can be generally used for gene therapy, such as adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, herpes virus vectors, vaccinia viruses. A vector, a retrovirus vector, etc. can be mentioned, By using these viral vectors, it can administer efficiently.
[0019]
Moreover, the method of introduce | transducing the polynucleotide which codes ADAMTS-1 protein or its functional equivalent modified substance into phospholipid vesicles, such as a liposome, and administering the liposome is also employable. That is, since the liposome is a closed vesicle containing a biologically degradable material, the polynucleotide is held in the aqueous layer or lipid bilayer inside the liposome by mixing the liposome and the polynucleotide. (Liposome-gene complex). Next, when the complex is cultured with cells, the gene in the complex is taken into the cells (lipofection method). The obtained cells can be administered into the body by the following administration method.
[0020]
The administration method is not particularly limited as long as it is administered in such a way that an effective amount reaches the affected area. For example, systemic administration (for example, intravenous administration, intraarterial administration, subcutaneous administration, intramuscular administration) Administration, or oral administration, etc.), topical administration, transmucosal administration, or intestinal administration. Furthermore, an administration method combined with catheter technique or surgical operation can also be adopted.
[0021]
The dose of the cancer metastasis inhibitor of the present invention can be appropriately determined depending on, for example, age, sex, symptom, administration route, number of administrations, dosage form, etc. Usually, ADAMTS-1 protein or functional thereof The range of 0.1-100 mg / individual per adult is appropriate for the weight of the polynucleotide encoding the equivalent variant.
[0022]
Examples of cancers that can suppress metastasis by the cancer metastasis inhibitor of the present invention include, for example, gastric cancer, colon cancer, liver cancer, kidney cancer, breast cancer, oral cancer, pancreatic cancer, esophageal cancer, bladder cancer, uterine cancer, or Examples include lung cancer.
As a mechanism in which the cancer metastasis inhibitor of the present invention exhibits a cancer metastasis inhibitory action, ADAMTS-1 protein contained as an active ingredient or a functionally equivalent variant thereof itself, or ADAMTS-1 protein contained as an active ingredient or It is considered that ADAMTS-1 protein produced in the body or a functional equivalent variant thereof is involved in suppression of cancer metastasis from a polynucleotide encoding the functional equivalent variant or a recombinant vector containing the polynucleotide. However, details are unknown at this stage.
[0023]
As shown in Example 2 described later, when the ADAMTS-1 gene is introduced into a cultured cell derived from lung cancer in a mouse experimental lung metastasis system, the metastasis to the lung is suppressed depending on the expression level of the ADAMTS-1 gene. It was done. Considering that ADAMTS-1 protein is an extracellular matrix-bound MMP, as described above in the prior art section, the ADAMTS-1 gene introduced into the cultured cells is expressed, and the extracellular of the cultured cells is expressed. ADAMTS-1 protein is produced. In Example 2, it is considered that this extracellular ADAMTS-1 protein suppressed cancer metastasis to the lung. Therefore, as shown in Example 2, without introducing the ADAMTS-1 gene into the target cancer cell that is the target of suppression of cancer metastasis, for example, by administering the ADAMTS-1 protein itself into the body, Alternatively, administration of a polynucleotide encoding ADAMTS-1 protein or a recombinant vector containing the same in the body to produce ADAMTS-1 protein in the body can suppress cancer metastasis as in Example 2. it can.
[0024]
【Example】
EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
[Example 1]
<< Preparation of ADAMTS-1 transgenic cancer cells >>
Mouse ADAMTS-1 expression vector (J. Biol. Chem., 273, 13912-13917, 1998) or control vector [pcDNA3; Invitrogen (San Diego, Calif., USA)], each in an amount of 5 μg, Mouse Lewis lung cancer strain (LLC; ATCC CRL-1642) was transfected with a commercially available transfection reagent [lipofectin; Life Technologies (Gaithersburg, Md., USA)]. After 24 hours, G-418 [Life Technologies (Gaithersburg, Md., USA)] was added to a final concentration of 200 μg / mL, followed by culturing. After 3 weeks, G-418 resistant transfectants were isolated and cultured.
[0025]
Total RNA was extracted and purified by a conventional method from seven types of ADAMTS-1 gene-introduced LLC transfectants and control vector-introduced LLC transfectants. Each 10 μg of RNA was subjected to denaturing agarose gel electrophoresis, and then the RNA was transferred onto a nylon (registered trademark) membrane. The membrane was treated under vacuum at 80 ° C. for 2 hours, then hybridized with ³² P-labeled mouse ADAMTS-1 cDNA (probe), washed, and autoradiographed on X-ray film. I did it. Thereafter, the probe on the membrane was removed by hot water treatment, and further hybridized with human β-actin cDNA, and autoradiography was performed in the same manner as described above.
[0026]
As a result of this Northern hybridization, transfectants LLC / ADAMTS-1 / C-13 that weakly express the ADAMTS-1 gene and transfectants LLC / ADAMTS-1 that strongly express the ADAMTS-1 gene. / C-12 and control vector-introduced transfectant LLC / Vector were selected and used for the following lung metastasis experiments of mice. FIG. 1 shows the northern hybridization results for the three selected transfectants.
[0027]
[Example 2]
《Inhibition of experimental lung metastasis in mice》
1 × 10 ⁶ LLC / ADAMTS-1 / C-12, LLC / ADAMTS-1 / C-13, and LLC / vector suspended in 200 μL of physiological saline, 7, 7, and Eight C57BL / 6 mice (9 weeks old) were injected into the tail vein and then sacrificed on day 14 to calculate the number of lung metastatic nodules.
[0028]
The results (mean value ± standard error) are shown in FIG. As shown in FIG. 2, LLC / ADAMTS-1 / C-12, which strongly expresses ADAMTS-1 gene, is significantly (p <0.034) metastasized compared to LLC / Vector (control). It was suppressed. Although LLC / ADAMTS-1 / C-13 expressing the ADAMTS-1 gene weakly had no significant difference, metastasis was suppressed from the control. From these results, it was found that LLCs into which ADAMTS-1 had been introduced were significantly suppressed in lung metastasis compared to controls. Moreover, the degree of suppression showed a positive correlation with the expression level of the ADAMTS-1 gene. From this, it was found that ADAMTS-1 protein is useful as an antimetastatic agent for cancer.
[0029]
【The invention's effect】
According to the cancer metastasis inhibitor of the present invention, cancer metastasis can be suppressed.
[Brief description of the drawings]
FIG. 1 is a photograph replacing a drawing, showing the results of Northern hybridization in a mouse Lewis lung cancer strain into which a mouse ADAMTS-1 gene was introduced.
FIG. 2 is a graph showing the results of suppression of lung metastasis of mouse ADAMTS-1 gene-introduced mouse Lewis lung cancer strain.

Claims (1)

A cancer metastasis inhibitor comprising, as an active ingredient, an ADAMTS-1 protein or a functional equivalent variant thereof, a polynucleotide encoding the ADAMTS-1 protein or a functional equivalent variant thereof, or a recombinant vector containing the polynucleotide.

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