JP2789682B2 - Novel polypeptides and their uses - Google Patents

Description

The present invention relates to a novel cancer metastasis inhibitor.

Surgical treatment, radiation, and chemotherapy are mainly used to treat cancer, but satisfactory therapeutic effects have not been achieved in terms of recurrence and metastasis of cancer. Many anticancer agents currently used are those that inhibit the biosynthetic system of nucleic acids and proteins and cause cancer cells to die. However, the effects of these anticancer drugs are indistinguishable from normal cells and cancer cells, and have a serious problem in terms of side effects. In addition, these anticancer drugs reduce the primary cancer site and treat it.However, the always problem in cancer treatment is that the cancer leaves the primary site, metastasizes to other organs, grows there, and has a fatal result. It is to invite. Therefore, for the fundamental treatment of cancer, there is a strong demand for the development of an anticancer agent which has an inhibitory effect on cancer cell proliferation and cancer metastasis and invasion as well as a cancer cell growth inhibitory effect.

With regard to the mechanism of cancer metastasis, many studies have been made and the search for substances related to the inhibition of metastasis has been widely performed. After being released from the primary focus, cancer cells invade blood vessels. The cancer cells also penetrate into the vascular endothelial cell layer of the blood vessel wall and adhere to the extracellular matrix component, and then destroy the extracellular matrix with a degrading enzyme. In this way, cancer cells are thought to invade other organs and form new metastases there (LALiotta, e
tal . (1983) Lab . Invest ., 49, 636-649). In order to develop a cancer metastasis inhibitor, it is considered that a drug that suppresses any of the above nuclear steps should be developed. For example, those that efficiently inhibit the binding between extracellular matrix and cells (eg, MJHumphries, K. Olden, and KMYamada (1986) Sc
ience , 223 , 467-470,) or Y. Iwamoto et al, Sc.
ience (1987) 238 , 1132-1134), substances that inhibit infiltration into vascular endothelial cells, substances that inhibit degradative enzymes (eg, T. Irimura, M. Nakajima, and GL Nicolson, (198
6) Biochemistry , 25, 5322-5328) and the like. Among them, substances having particularly weak side effects are desirable.

The present inventors have conducted intensive studies on a substance that suppresses cancer cell invasion as one of cancer metastasis inhibitors, and as a result, using ion exchange method, gel permeation method, reverse phase column method and the like from mammalian liver, We succeeded in isolating and purifying the following polypeptides, which have a strong inhibitory effect on cancer cell invasion, and completed this study.

(However, Glx represents Glu or Gln) When numbering each amino acid residue from the N-terminus to the C-terminus as described above, the tenth and twelfth amino acid residues are Glu and Gln. Either may be used. However, preferably, the polypeptide is a polypeptide in which the tenth is Gln and the twelfth is Glu, and a polypeptide in which the tenth is Glu and the twelfth is Gln.

An experimental cancer metastasis system using B16 melanoma, Lewis lung cancer cells and the like is widely used as an evaluation system for developing antimetastatic agents. However, the results of these screening methods vary widely, and the number of drugs to be screened is limited, so that this method is not necessarily a preferable method for screening a large number of cancer metastasis inhibitors. In the present invention, as a method for searching for a substance having an inhibitory effect on cancer cell invasion, a method developed by Akada et al . ( Cancer Res.
6) Screening was performed using 46 , 2416-2422).
The results of the in vitro test method by Akira et al. Show a very good correlation with the in vivo test, and are considered to be excellent as screening methods for cancer metastasis inhibitors. According to the method of Akada et al., A primary cell layer is prepared by performing primary culture, a floating cancer cell is added thereto, incubated for a while (20-40 hours), and then sunk under the cell layer. This is a method for measuring the number of cancer cells. Normal cells include mesenteric mesothelial cells and vascular endothelial cells. There are many types of cancer cells, for example, ascites hepatoma cell AH-130 and the like. According to this method, many substances were tested for their metastatic inhibitory effect.

Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.

Example 1 Purification of Cancer Cell Invasion Inhibitor from Liver Bovine liver (5 kg) was treated with hydrochloric acid (187.5 ml) and ethanol (9.
5l) and homogenized with a mixed solution of PMSF (1 g), and the solution was stored at 4 ° C. overnight to perform extraction. Thereafter, the mixture was separated into a supernatant and a sediment by a centrifugation operation (7,500 rpm, 60 minutes). Aqueous ammonia was added to the supernatant to adjust the pH to 5.2, and 1/85 volume of 2M ammonium acetate (pH 5.2) was further added. After mixing at 4 ° C. overnight, the supernatant and the sediment were separated by centrifugation (9000 rpm, 30 minutes). The supernatant (250 ml) was cooled with ether (500 ml).
l), a mixture of ethanol (250 ml) was added,
A precipitate formed overnight. The resulting precipitate was dissolved in 1M acetic acid, dialyzed against 0.17M acetic acid, sufficiently dialyzed, freeze-dried, and further dissolved in physiological saline. The resulting solution was filtered through a membrane filter (Diaflo UM-10) and collected.

A DEAE-Toyopearl column (5 cm) equilibrated with the solution obtained as described above with 10 mM ammonium acetate (pH 7.2)
× 12 cm) and eluted with a concentration gradient with OM-0.2M NaCl (fraction graph is shown in FIG. 1). Active fractions (bars in Fig. 1) were collected, lyophilized, and
Gel filtration was performed on an el P-6 column (1 × 100 cm) (fraction graph is shown in FIG. 2). 0.1M acetic acid was used as the eluate, and the active fractions corresponding to the molecular weights of 2,000 to 3,000 (bars in FIG. 2) were collected, and were collected on an ODS column (YMC-PACK, D-ODS-
5) Fractionation (2 × 25 cm) (fraction graph is shown in FIG. 3), and the active fraction (bars in FIG. 3) was further applied to an ODS-column (Waters μ BONDASPHERE) (fraction graph). Are shown in FIG. 4), and active fractions (bars in FIG. 4) were collected.
As a result, the obtained active fraction was identified by the OD shown in FIG.
In the same test as in the S-column fractionation, as shown in FIG. 5, the polypeptide was highly pure and had an inhibitory effect on cancer cell invasion.

By the above purification method, a total of about 5 μg of the purified polypeptide was obtained from 100 kg of the liver.

Identification of polypeptide Amino acid sequence from the N-terminus was determined using 1 μg of the purified polypeptide. The procedure was performed according to a conventional method using a protein sequencer 477A-120A manufactured by Appleid Biosystems. As a result, it was found that the polypeptide was composed of 21 amino acids as shown below.

In addition, It was also found that there was a polypeptide having the same sequence as the following except that This result was consistent with the number of each amino acid obtained from the separately analyzed amino acid analysis value, and thus it was confirmed that the structure was a full-length structure. In addition, when the above two polypeptides were chemically synthesized and their effects on cancer cells were measured as described below, both had the same activity.

Example 2 Effect of the present substance on infiltration of ascites hepatoma cell AH-130-clone 30 cells The evaluation method was performed according to the method of Akado et al. That is, 5-7 days after primary culture of rat mesenteric cells, ascites hepatoma cells AH130-clone 30 were added to form a single cell sheet, and after 24 hours, the number of hepatoma cells infiltrating under the cell sheet was measured. I do. According to this method, the invasion inhibitory activity of the polypeptide obtained in Example 1 was measured. The results are shown in FIG. FIG. 6 shows the infiltration inhibition ratio (%) (vertical axis) with respect to the concentration of the present polypeptide (horizontal axis). As shown in the figure, the polypeptide was shown to have an effect of inhibiting invasion of cancer cells.

[Brief description of the drawings]

FIGS. 1 to 5 are graphs showing the results of fractionation by column chromatography in the purification of the active ingredient of Example 1, and the bars in the graphs of FIGS. 1 to 4 indicate the presence of the active ingredient. Indicates the area. FIG. 5 shows the final purification results. FIG. 6 is a graph showing evaluation results of Example 2. The columns used in FIGS. 1 to 5 are as follows. Figure 1: DETA-Toyo pearl column Figure 2: Biogel P-6 column Figure 3: ODS-column Figure 4: ODS-column Figure 5: ODS-column (same as Figure 4)

──────────────────────────────────────────────────続 き Continuation of the front page (56) References European Journal of Biochemistry, Vol. 76, p. 46-468 (1977) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14/47 A61K 37/02 A61K 35/407 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A novel polypeptide having the following sequence: (However, Glx represents Glu or Gln) 2. A cancer metastasis inhibitor comprising the novel polypeptide according to claim 1 as an active ingredient.