JPH0358360B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a cancer cell proliferation inhibitor, which is a novel protein-based substance that is separated from human blood and serum and has a malignant tumor cell proliferation inhibitory effect.

The present inventors isolated various proteins from human living bodies as substances that suppress the proliferation of malignant tumor cells.
As a result of our research, we discovered that a protein isolated from blood has the effect of suppressing the proliferation of various malignant tumor cells in vitro and has the effect of prolonging the survival of animals inoculated with malignant tumor cells in vivo.

The protein is a protein named cancer cell growth inhibitory factor (TDFH) by the present inventors due to its chemical, physical, and biological properties, and is obtained from human blood and serum. It has the effect of suppressing the proliferation of malignant tumor cells and shows a remarkable survival effect on animals transplanted with malignant tumor cells. In particular, although it has a strong growth-suppressing effect on malignant tumor cells, it does not show any malignant tumor cell-killing effect in vitro, is non-toxic to humans or animals, and does not show any observable inhibitory effect on normal cells. It should be said that it has specific physiological activity in this respect, and is extremely valuable as a medicine.

The above-mentioned protein cancer cell growth inhibitory factor [TDF
H] is obtained from human blood and serum by salting out, extraction, adsorption, elution, dialysis, fractionation, precipitation, filtration, isoelectric precipitation, gel filtration, use of ion exchange resin, electrophoresis, etc. based on its properties. Separation and acquisition can be achieved by appropriately combining known operations.

More specifically, first, ammonium sulfate, salt, acetone, alcohol, etc. are added to blood or serum to precipitate the active ingredient, and this precipitate is dialyzed and dried to obtain a coarse powder of cancer cell growth inhibitory factor [TDFH]. .

For further purification, column chromatography using ion exchange resins, Cephadex, activated carbon, silica gel or diethylaminoethyl (abbreviated as DEAE) and cellulose, and electrophoresis or isoelectric precipitation using polyacrylamide gels, cellulose, etc. Furthermore, cancer cell growth inhibitory factor [TDFH] can be purified by adsorbing it on an adsorbent such as silica gel, bentonite, acid clay, or activated carbon.

The protein substance cancer cell proliferation inhibitory factor [TDFH] isolated from human blood and serum as described above exhibits extremely specific physiological effects as described above and is useful as an anti-malignant tumor agent.

As a result of further research into this protein, cancer cell growth inhibitory factor [TDFH], the present inventors discovered that it is not a single substance, but can be further separated into several components, and that all of these components are associated with malignant tumor cells. Knowing that they have a growth-suppressing effect, we have added these to cancer cell growth-suppressing factors A, B, C [TDFH...A, B,
It was named C.

Among these, cancer cell growth inhibitory factor B [TDF
HB] is a novel cancer cell proliferation inhibitor according to the present invention.

That is, during the above-mentioned salting out, the alcohol saturation degree is varied, for example, 40%, 50%, 60%, and fractional precipitation is carried out according to the molecular weight, adsorption with a molecular sieve,
After increasing the purity through elution, careful fractionation is performed using column chromatography to obtain cancer cell growth inhibitory factor B [TDFHB] of the present invention from a mixture of cancer cell growth inhibitory factors A, B, and C. I can do it. For reference, the physical properties of cancer cell proliferation inhibitory factors A and C are also described below.

Cancer cell growth inhibitory factor A [TDFHA] Appearance: White powder Melting point: 245-259℃ Decomposition properties: Glycoprotein molecular weight: Approximately 73,900 (by 10% sodium dodecyl sulfate electrophoresis) Solubility: Easily soluble in water, butanol, acetone , ethyl acetate, chloroform, benzene, hexane-insoluble amino acid composition: aspartic acid 10.0%, threonine 5.4%, serine 3.8%, glutamic acid 16.0%,
Proline 4.2%, glycine 1.4%, alanine 4.8
%, cystine 2.6%, valine 5.1%, methionine
0.8%, isoleucine 2.2%, leucine 9.6%, tyrosine 4.9%, phenylalanine 5.7%, lysine 11.4%, histidine 3.5%, arginine 5.2% Color reaction: Ninhydrin, Anthrone, Mauritsch, periodic acid reaction positive infrared absorption spectrum :See Figure 1 [measured with KBr pellets] Ultraviolet absorption spectrum: Figures 2, 3, and 4
See figure Maximum absorption at 279 nm in PH3.0 water (see Figure 2) Maximum absorption at 278 nm in PH7.0 water (see Figure 3) Maximum absorption at 277 nm in PH10.0 water (see Figure 4) Cancer cell growth Inhibitor factor B [TDFHB] Appearance: White powder Properties: Protein Melting point: 221-225℃ Degraded molecular weight: Approximately 22,100 (by 10% sodium dodecyl sulfate electrophoresis) Solubility: Easily soluble in water; acetone, ethyl acetate, benzene, Amino acid composition insoluble in hexane and chloroform: aspartic acid 16.0%, threonine 5.2%, serine 5.8%, glutamic acid 6.9%, proline 2.3%, glycine 4.1%, alanine 5.9%,
Cystine 2.3%, valine 4.4%, methionine 1.7
%, Isoleucine 4.2%, Leucine 7.9%, Tyrosine 4.0%, Phenylalanine 3.9%, Lysine
7.7%, histidine 1.6%, arginine 11.6% Color reaction: positive ninhydrin reaction, Anthrone,
Mauritius, periodic acid reaction negative infrared absorption spectrum: See Figure 5 [measured with KBr pellets] Ultraviolet absorption spectrum: Maximum absorption at 283 nm and 290 nm in PH3.0 water (see Figure 6) Maximum at 280 nm in PH7.0 water Absorption (see Figure 7) Maximum absorption at 277 nm in PH10.0 water (see Figure 7)
(See figure) Malignant tumor cell growth inhibitory factor C [TDFHC] Appearance: White powder Melting point: 237-240℃ Decomposition properties: Protein molecular weight: Approximately 14,000 (by 10% sodium dodecyl sulfate electrophoresis) Solubility: Easily soluble in water; Insoluble in acetone, ethyl acetate, benzene, hexane, chloroform Amino acid composition: aspartic acid 18.0%, threonine 5.4%, serine 6.4%, glutamic acid 5.2%, proline 1.7%, glycine 5.1%, alanine 6.4%,
Cysteine 2.1%, Valine 4.3%, Methionine 1.9
%, isoleucine 4.5%, leucine 6.8%, tyrosine 3.6%, phenylalanine 3.0%, lysine
6.0%, histidine 1.1%, arginine 11.6% Color reaction: ninhydrin reaction positive, Anthrone,
Mauritius, periodic acid reaction negative Infrared absorption spectrum: See Figure 9 [measured with KBr pellets] Ultraviolet absorption spectrum: 278 nm in PH3.0 water
(See Figure 10) Absorption maximum at 290 nm in PH 7.0 water (See Figure 11) Absorption maximum at 277 nm in PH 10.0 water (1st
(See Figure 2) Cancer cell growth inhibitory factor A, cancer cell growth inhibitory factor B, and cancer cell growth inhibitory factor C described above were PH8.0, PH9.5, PH by 7% acrylamide gel electrophoresis.
In 10.0, they all move from the origin and show a single band.

Therefore, instead of chromatography, it is also possible to perform fractionation by acrylamide gel electrophoresis. Cancer cell growth inhibitory factor A obtained by such a purification method
[TDFHA], cancer cell growth inhibitory factor B [TDFH]
B], cancer cell growth inhibitory factor C [TDFHC], as well as the coarse powder obtained at each stage can be used as various malignant tumor cell growth inhibitors.

As mentioned above, even in pure form without adding other ingredients,
Alternatively, it is effective in the form of a mixture, and can be prepared into injections and other various dosage forms.

Furthermore, the cancer cell proliferation inhibitory factor B [TDF
Reference examples are also shown for cancer cell proliferation inhibitory factors A and C, which are obtained as a mixture in crude purification.

The present invention will be explained below with reference to Examples.

Reference example 1 Add twice the amount of acetone to human serum 1 at -20℃, centrifuge the precipitate, dissolve it in the minimum amount of distilled water, and add 45 to 50% ethyl alcohol.
8.6 g of fractionated precipitate was collected.

5g of it at PH7.3 and 20% of Cephadex G200
ml volume and eluted with 30 ml of 0.05M Tris-HCl buffer at pH 7.3 to obtain 0.9 g of a crude substance of cancer cell growth inhibitory factor A.

Next, diethylaminoethyl (DEAE) was adsorbed on cellulose (manufactured by Watmann), and the salt concentration was adjusted.
Elute with 40 ml of 0M to 0.5M salt gradient solution,
Divide into 7 major fractions. The fifth fraction was dialyzed against 0.02M Tris buffer, desalted, and lyophilized to obtain 23.7 mg of crude material.

Collect 50mg of this substance, adsorb it on diethylaminoethyl (DEAE) cellulose (manufactured by Watmann), and adjust the pH from PH8.0 to PH5.0 with 0.1M salt solution.
Elution was performed using an ion concentration gradient, and the second fraction, which was separated into five fractions, was desalted and lyophilized to obtain 7.3 mg of cancer cell growth inhibitory factor A.

This was electrophoresed on a 7% acrylamide gel for 4 hours, and the ninhydrin-positive second band
After elution with 0.02M Tris buffer, dialysis, and drying,
Cancer cell proliferation inhibitory factor A [TDFHA] showing a single band in electrophoresis of 0.6 mg was obtained.

At 0.01 mg/ml, this substance was effective in suppressing the proliferation of Leukemia L1210 cells in tissue culture by 99%. Furthermore, it showed an inhibitory activity of 61.5% at 0.001 mg/ml.

Reference Example 2 31.2 mg of the fifth fraction obtained from the first diethylaminoethyl (DEAE) cellulose (Watmann) chromatography using the same procedure as Reference Example 1.
The active ingredient was isolated as a single substance using acrylamide gel electrophoresis under pH 9.5 conditions, dialyzed, and freeze-dried to produce 0.6 mg of purified cancer cell growth inhibitory factor A as a white powder. Obtained.

Example 1 Add 60% alcohol to human serum 8, remove the precipitate, add alcohol to 70%, and adsorb the resulting precipitate in 40 ml of Cephadex G-50 at pH 7.3. Seshime, 0.05M of PH7.3
Elution was performed with 50 ml of Tris-HCl buffer to obtain 5.8 g of a crude substance of cancer cell growth inhibitory factor B.

Next, it was adsorbed on diethylaminoethyl (DEAE) cellulose (manufactured by Watmann), and 0.02M ~
Elute with 50 ml of 0.05M Tris-HCl buffer (PH7.3) ionic strength gradient solution and divide into 6 major fractions.

The first fraction was dialyzed, desalted, and lyophilized to obtain 25.4 mg of crude material.

This is further converted into diethylaminoethyl (DEAE)
Adsorbed with cellulose (manufactured by Watmann),
Elute with PH ion concentration gradient from PH8.0 to PH5.0 with 0.1M saline solution and divide into 6 fractions.The first fraction is dialyzed, desalted, and freeze-dried to produce white cancer cell growth inhibitory factor. 4.2 mg of B [TDFHB] was obtained.

At 400 mcg/ml, this substance exhibited activity that delayed the growth curve angle of Leukemia L1210 cells in tissue culture by approximately 20 degrees, and showed a single band in electrophoresis.

Example 2 Using human serum 2, the alcohol precipitate shown in Example 3 was adsorbed on diethylaminoethyl (DEAE) cellulose (manufactured by Watmann), and the mixture was adjusted to pH 8.0 to PH 5.0 with 0.1M saline solution. The first fraction was eluted with a pH ion concentration gradient and divided into 6 fractions.The first fraction was dialyzed, desalted, and lyophilized.The active ingredient was analyzed as a single substance by electrophoresis using a 7% polyacrylamide gel. After fractionation, dialysis, and lyophilization,
85mcg of cancer cell growth inhibitory factor B [TDFHB]
I got it.

This product showed the same activity in suppressing the Leukemia L-1210 cell growth curve angle in tissue culture as in Example 1.

Reference Example 3 Add 70% alcohol to human serum 8, remove the precipitate, and concentrate the resulting supernatant under reduced pressure.
This was further freeze-dried, adsorbed onto 40 ml of Cephadex G-50 at pH 7.3, and eluted with 30 ml of 0.05M Tris-HCl buffer at pH 7.3. Approximately 1.0 g of the crude substance of cancer cell proliferation inhibitory factor C [TDFHC] was obtained. I got it.

Next, diethylaminoethyl (DEAE) was adsorbed on cellulose (manufactured by Watmann), and
Elute with 50 ml of 0.05M Tris-HCl buffer (PH7.3) ionic strength gradient solution and divide into three major fractions.

The second fraction was dialyzed, desalted and lyophilized to yield 14.5 mg of crude material.

These were further adsorbed on diethylaminoethyl (DEAE) cellulose (manufactured by Whattman), eluted with a PH ion concentration gradient from PH8.0 to 5.0 with 0.1M saline solution, and divided into 6 fractions. was dialyzed, desalted, and lyophilized to obtain 1.8 mg of white cancer cell growth inhibitory factor C [TDFHC].

This substance shows a single band in electrophoresis, and
Leukemia L1210 in tissue culture at mcg/ml
The activity showed that the cell growth curve angle was delayed by about 10 degrees.

Reference Example 4 A lyophilized supernatant obtained using human serum 2 in the same manner as in Example 5 was added to diethylaminoethyl (DEAE) cellulose (manufactured by Watmann).
It was adsorbed onto a 0.1M saline solution and eluted with a pH ion concentration gradient from PH8.0 to PH5.0.The fourth fraction was divided into 6 fractions, dialyzed, desalted, and after freeze-drying, polyacrylamide Using a gel, the active ingredient was fractionated as a single substance by electrophoresis, dialyzed, and freeze-dried, resulting in 184 mcg of cancer cell growth inhibitory factor C [TDF
HC] was obtained.

As shown in Example 5, this product exhibited the activity of suppressing the Leukemia L1210 cell growth curve angle in tissue culture.

Reference Example 5 Human serum-derived cancer cell growth inhibitory factor A500mcg/ml obtained in Reference Example 1 and Reference Example 2
We investigated the inhibitory effect on malignant tumor cell proliferation in tissue culture using the following methods.

It showed a growth-inhibiting effect of 32.6% against Leukemia L-5178Y cells, 21.3% against Sarcoma S-180 cells, and 32.3% against Ehrlitsu carcinoma cells, but it was effective against primary tissue culture cells of rat liver. showed no growth-inhibiting effect at all. Also,
The survival of the various malignant tumor cells tested was confirmed by staining tests.

Reference Example 6 Animal experiments were conducted using human serum-derived cancer cell proliferation inhibitory factor A [TDFHA] obtained in Reference Example 1.

A test animal group consisting of 5 BDF mice (male, weight 20.0 g) was used, respectively.

In the first group, cancer cell growth inhibitory factor A [TDFH
A] 0.1 mg/mouse, 1×10 ⁶ Leukemia L1210 cells
was injected intraperitoneally, followed by intraperitoneal injection 24 hours later.

In the second and third groups, the same experiment as in the first group was conducted, but the sample projection amount was 0.01 in the second group.
mg/mouse, and 0.001 mg/mouse in the third group.

Controls died after an average of 9.7 days, while group 1 died after an average of 9.7 days.
The second group died in an average of 15.7 days;
Furthermore, the third group died after an average of 12.3 days. From this, the T/C value was 207 for the first group, 162 for the second group, and 128 for the third group.

Example 3 In a test using 500 mcg/ml of human serum-derived cancer cell growth inhibitory factor B [TDFHB] obtained in Example 3, it was found to have an effect of about 15 degrees on Leukemia L5178Y cells in tissue culture and on Sarcoma S-180 cells. About 14 degrees for Ehritzhi carcinoma cells, about 5 degrees for Ehritzhi carcinoma cells
The cell proliferation curve showed activity indicating a lag in angle.

However, it did not show any growth-inhibiting effect on primary tissue culture cells derived from rat liver.
In addition, the survival of the various malignant tumor cells tested was confirmed by staining tests.

Reference Example 7 Human serum-derived cancer cell growth inhibitory factor C [TDFHC] 500m obtained in Reference Example 3 and Reference Example 4
In a test using cg/ml, Sarcoma S-
The activity showed a delay in the cell growth curve angle of about 22 degrees for 180 cells and 1 degree for Ehritzhi carcinoma cells.

However, it did not exhibit any growth-inhibiting effect on primary tissue cultures derived from rat liver. In addition, the survival of the various malignant tumor cells tested was confirmed by staining tests.

[Brief explanation of drawings]

FIG. 1 shows cancer cell proliferation inhibitory factor B [T.
Figures 2, 3, and 4 show the infrared absorption spectra of cancer cell growth inhibitory factor A [TDFHA], which is obtained as a mixture in crude purification, and cancer cell growth inhibitory factor A [TDFHA]. FIG. 5 shows the ultraviolet absorption spectrum at PH3.0, PH7.0 and PH10.0, respectively, and FIG. 5 shows the infrared absorption spectrum of cancer cell proliferation inhibitory factor B [TDFHB] of the present invention. Figure 8 shows PH3.0 and PH of cancer cell growth inhibitory factor B [TDFHB], respectively.
Ultraviolet absorption spectra at 7.0 and PH10.0,
Further, FIG. 9 shows the cancer cell proliferation inhibitory factor B of the present invention.
[TDFHB] and cancer cell growth inhibitory factor C [TDFHC] obtained as a mixture in crude purification.
The infrared absorption spectra of
The figure and FIG. 12 show the ultraviolet absorption spectra of cancer cell proliferation inhibitory factor C [TDFHC] at PH3.0, PH7.0, and PH10.0, respectively.

Claims (1)

[Claims] 1. A white powder with a melting point of 221 to 225°C (decomposed);
Molecular weight determined by 10% sodium dodecyl sulfate electrophoresis is approximately 22,100; readily soluble in water, insoluble in acetone, ethyl acetate, benzene, hexane, and chloroform; aspartic acid 16.0%, threonine
5.2%, serine 5.8%, glutamic acid 6.9%, proline 2.3%, glucine 4.1%, alanine 5.9%, cystine 2.3%, valine 4.4%, methionine 1.7%, isoleucine 4.2%, leucine 7.9%, tyrosine 4.0%, phenylalanine It has an amino acid composition of 3.9%, lysine 7.7%, histidine 1.6%, and arginine 11.6%; positive for ninhydrin reaction, negative for Anthrone, Mauritius, and periodic acid reactions, and exhibits the infrared absorption spectrum [KBr pellet] shown in Figure 5; Ultraviolet absorption spectrum is 283nm and 290n in water with PH3.0
m, 280nm in PH7.0 water, 277n in PH10.0 water
A protein named cancer cell growth inhibitory factor B [TDFHB], which shows absorption maximum at m, is separated from human blood and serum, and has malignant tumor cell growth inhibitory effect.