JPS6048931A - Tumor cell proliferation inhibiting factor consisting of novel protein - Google Patents

Abstract

NEW MATERIAL:The tumor cell proliferation inhibiting factor A (T.D.F.H.A) [melting point, 245-249 deg.C (decomposition); white powder; molecular weight, 73,900 (electrophoresis as 10% sodium dodecylsulfate); solubility, easily soluble in water and insoluble in butanol, acetone, etc.; amino acid composition, shown in the table; color reaction, positive to ninhydrin, anthrone, etc.]. USE:Substance for suppressing the proliferation of malignant tumor cell. PREPARATION:Ammonium sulfate, sodium chloride, acetone, alcohol, etc. are added to blood or blood serum to effect the precipitation of the active component. The precipitate is dialyzed and dried to obtain crude powder of T.D.F.H, which is purified by the ion exchange resin treatment and/or sephadex treatment, etc. The purified produce can be further purified by adsorbing to an adsorbent such as silica gel, bentonite, acidic clay, etc. It can be administered in the form of injection, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel protein-based substance that is separated from human blood and serum and has an inhibitory effect on the growth of malignant tumor cells.
It relates to cancer cell growth inhibitory factors.

The present inventors isolated and studied various proteins from human organisms as substances that suppress the proliferation of malignant tumor cells.
We have discovered that a protein isolated from blood has the effect of suppressing the proliferation of various acute tumor cells in vitro and has the effect of prolonging the survival of animals inoculated with malignant tumor cells in vivo.

(3) The present inventors have identified this protein as a cancer cell proliferation inhibitor (T, D,
This is a novel protein named F, II), which is obtained from human blood and serum, has an acute tumor cell proliferation inhibitory effect, and exhibits 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 and animals, and shows no appreciable inhibitory effect on normal cells. It should be said that it has specific physiological activities in several respects, and is extremely valuable as a medicine.

This novel protein cancer cell growth inhibitory factor (T, D, F, H) according to the present invention is extracted from human blood or serum by salting out, extracting 2g, depositing, and eluating based on its properties.

Dialysis 1 fractionation, ill precipitation, Oki 9 isoelectric precipitation, gel filtration, use of ion exchange resin, 1! Separation and acquisition can be achieved by appropriately combining known operations such as L-vaporphoresis.

More specifically, first, ammonium (4) sulfate, salt, acetone, alcohol, etc. are added to blood or serum to precipitate the active ingredients, and this precipitate is dried after precipitation to produce cancer cell growth inhibitory factors (T, D, F,) A coarse powder of I) is obtained.

For further purification, column chromatography using ion exchange resins, Sephadex, activated carbon, silica gel or diethylaminoethyl (abbreviated as DEAE) and cellulose, and electrophoresis using polyacrylamide gels, saccharides, etc. This is possible by using point precipitation alone or in combination, and furthermore, cancer cell proliferation inhibitory factors (T, D, F, H), silica gel,
It can be purified by adsorption onto an adsorbent such as bentonite, acid clay, or activated carbon.

The protein substances cancer cell growth inhibitory factors (T, D, F, H) isolated from human blood and serum as described above exhibit extremely specific physiological actions as described above, and are useful as anti-malignant tumor agents. It is.

As a result of further research into this novel protein cancer cell proliferation inhibitor (T, D, F, H), the present inventors discovered that it is not a single substance but can be further separated into several components. , we learned that all of them have the effect of suppressing the growth of malignant tumor cells, and we decided to use cancer cell growth suppressors A, B, and C (T, D).

F, H...A, B, C).

That is, during the above-mentioned salting out, the alcohol saturation is varied, for example, 40%, 50%, 60%, and fractional precipitation is carried out according to the molecular weight, purity is increased by adsorption and elution with a molecular sieve, and then column chromatography is carried out. When carefully separated, the following cancer cell growth inhibitory factors A, B, and C can be detected.
(T, D, F...A, B and C] are obtained.

Cancer cell growth inhibitory factor A (T, D, F, H, A) Appearance: White powder Melting point: 245-259°C Degradation properties: Glycoprotein molecular weight: Approximately 73,900 (by 10% sodium dodecyl sulfate electrophoresis) Solubility : Easily soluble in water, insoluble in butanol, acetone, ethyl acetate, chloroform, benzene, hexane 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, Molish.

Periodic acid reaction positive red IS line absorption spectrum: See Figure 1 (measured with KBr pellets) Ultraviolet absorption spectrum: See Figures 2, 3, and 4 Absorption maximum at 279 nm in PH3.0 water (see Figure 2) (See figure) (7) Maximum absorption at 278 am in pH 7.0 water (see Figure 3) Maximum absorption at 277 am in pH 10.0 water (see Figure 4) Cancer cell growth inhibitory factor B ('r, D, F,
H, B) Appearance: White powder Properties: Protein melting point = 221-225°C Decomposition molecular weight: Approximately 22,100 (by 10% sodium dodecyl sulfate electrophoresis) Solubility: Easily soluble in water, polyacetone, ethyl acetate, benzene,
Amino acid composition insoluble in hexane and chloroform: Aspartic acid 16. O%? Threonine 5.2%, Serine 5.8%, Glutamic acid 6.9%, Proline 2.3%
, glycine 4.1%, γlanin 5.9%, cystine 2.
3%, valine 4.4%, methionine 1.7%, isoleucine 4.2%, leucine 7.9%, tyrosine 4.0%,
Phenylalani(8) 3.9%, lysine 7.7%, histidine 1.6%,
Arginine 11.6% Color reaction: ninhydrin reaction, Anthrone, Molish,
Periodic acid reaction negative infrared absorption spectrum: See Figure 5 (measured with KBr pellets) Violet I11 absorption spectrum: Maximum absorption at 2B3nWP and 290aia in PH3,0 water (see Figure 6) P R'7,0 water Maximum absorption at 280218 in water (see Figure 7) P11]0.(+ Maximum absorption at 2774 in water (
(See Figure 8) Malignant tumor cell growth inhibitory factor c (: T, D, F, H, C
) Appearance: White powder Melting point = 237-240℃ Decomposition properties: Protein molecule M: Approximately 140 (10 (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 18.0%, Threonine 5.4%, Serine 6.4%, Glutamine #5.2%, Proline 1.7
%, glycine 5.1%, alanine 6.4%, cysteine 2.1%, valine 4.3%, methionine 1.9%, isoleicine 4.5%, leucine 6.8%, tyrosine 3
．． 6%, phenylalanine 3.0%, lysine 6. θ%
, histidine 1.1%, arginine 11.6% Color reactions: positive for ninhydrin, negative for Anthrone, Molish, periodic acid, infrared tia@according Specke spectrum diagram (measured with KBr pellets), ultraviolet Is@according to Sui Spectrum H3,0 278 turtle attacks underwater (see Figure 10) PH7,
Maximum absorption at 290 mm in 0 water (see Figure 11) P) 11 (maximum absorption at 2778 mm in 1,0 water (see Figure 11)
(See Figure 12) Cancer cell growth inhibitory factor A, cancer cell growth inhibitory factor B, and cancer cell growth inhibitory factor C described above were determined by 7% acrylamide gel 1ki pneumophoresis at pH 8.0, pH 9°5, Pl.
At i 10.0, the ψ shift also moves from the origin and shows a single band.

Therefore, instead of chromatography, it is also possible to perform fractionation by acrylamide gel electrophoresis. Cancer cell proliferation inhibitory factor A (T, D, F) obtained by such a purification method
, H, A), cancer cell growth inhibitory factor B (T.

D, F, summer 1. B), cancer cell growth inhibitory factor C (T, D,
F, H, C) Of course, the coarse powders obtained at each stage can also be used as various malignant tumor cell proliferation inhibitors.

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

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

(11) Example 1 Two volumes of acetone were added to human serum 11 at -20°C, the precipitate was centrifuged and dissolved in distilled water with a minimum lid, and then 45 to 50 ml of ethyl alcohol was added. A fractionated precipitate of 8°6f was collected.

Of that, 5f is PH7.3 and Sephadex G200 2
It was adsorbed to a volume of 0 ml and eluted with 30 zt of 0.05M Lis-HCl buffer (pH 7.3) to obtain 0.91 of a crude substance of cancer cell growth inhibitory factor A.

Next, diethylaminoethyl (DEAE) was adsorbed on cellulose (Whatman), and the salt concentration was adjusted to 0.5 from OM.
Elute with 40 ml of a sodium chloride concentration gradient solution of M and divide into 7 major fractions. The fifth fraction was dialyzed against 0.02M 1JS buffer, desalted, and lyophilized to yield 23.7 caps of crude material.

Collect 50 capes of this stuff and use diethylaminoethyl (DEA).
E) Adsorbed on cellulose (manufactured by Whatman) and 0.
It was eluted with a PR ion concentration gradient from PH8.0 to PH5.0 with 1M saline solution and fractionated into 5 fractions.02) The second fraction was desalted and lyophilized, and the 7.3q cancer cell growth inhibitory factor was detected. I got an A.

This was electrophoresed on a 7% acrylamide gel for 4 hours.
0.02M from the ninhydrin positive second band)! After elution with JS buffer, dialysis, and drying, cancer cell growth inhibitory factor A (T, D
, v, H, A) were obtained.

This substance showed the effect of suppressing the proliferation of Leukemia L 1210 cells in tissue culture by 99% at 0°01q/,/. Furthermore, it showed an inhibitory activity of 61.5% at 0.001q/gl.

Example 2 In the same manner as in Example 1, the fifth fraction 31.21v obtained from the first diethylaminoethyl (DEAE) cellulose (Whatman) chromatography was subjected to acrylamide gel electrolysis under the condition of pH 9.5. When the active ingredient was separated as a single substance by electrophoresis, dialyzed, and freeze-dried,
Cancer cell proliferation inhibitory factor A purified as a white powder of 0.11+y was obtained.

Example 3 h) Add 60% alcohol to serum 8#, remove the precipitate, add alcohol to 70%, and add the obtained precipitate to 40% Sephadex G-50 at pH 7.3. 0.05M with pH 7.3.
) Elution was carried out with 50 zt of Lis-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 Whatman), and 0.02M to 0.05
Elute with 50 yd of Tris-HCl buffer (PH7.3) ionic strength gradient solution and divide into 6 major fractions.

The crude substance 2 was dialyzed for 541 minutes, desalted, and lyophilized.
Obtained 5.4 capes.

This was further adsorbed on diethylaminoethyl (DEAE) Cell 4's (manufactured by Whatman), eluted with a PH ion concentration gradient from PH8.0 to PH5.0 with 0.1M salt solution, and divided into 6 fractions. The first fraction is dialyzed and desalted;
When freeze-dried, cancer cell growth inhibitory factor B (T, D,
F, H, B) 4.2W was obtained.

At 400 whef/liter, this substance showed activity showing a delay in the growth curve angle of about 204 for quaternary Ikemya L 1210 cells in tissue culture, and showed a single band in electrophoresis.

Example 4 Using human serum 21, the alcohol precipitate shown in Example 3 was adsorbed on diethylaminoethyl (DEjl) cellulose (manufactured by Whatman), and the pH was adjusted to PH 8, θ to PH 5, 0 with 0.1 M saline solution. The first fraction was eluted with a pH ion concentration gradient of After dialysis and freeze-drying, 85 cg of cancer cell growth inhibitory factor B (T,
D.F.

H, B:) was obtained.

This product showed the same activity in suppressing the Leukemia L-1210 cell growth curve angle in tissue culture as in Example 3.

Example 5 70% alcohol was added to human serum 81, the resulting precipitate was removed, and the resulting supernatant was concentrated under reduced pressure.05) It was further freeze-dried, and this was transferred to Sephadex G- Adsorbed to 40m of 50, 0.0 of PH7.3
The crude substance of cancer cell growth inhibitory factor C (T, D, F, H, C) was eluted with 5M) IJS hydrochloric acid buffer at 30xt. I got Oy.

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

#! The two fractions were dialyzed, desalted and lyophilized to yield crude material 14
．． 51M was obtained.◇ This was further adsorbed on diethylaminoethyl (DEAE) cellulose (manufactured by Von Co., Ltd.), and eluted with a 0.1M salt solution using a pH ion concentration gradient from PH8.0 to 5.0. Divided into 6 fractions, dialyzed and desalted the 4th fraction,
When freeze-dried, white cancer cell growth inhibitory factor C (T, D
, F, H, C) 1.8 capes were obtained.

This substance showed a single band in electrophoresis, and 50006) Leukemia L 12 in tissue culture at vhcy/ml.
The growth curve angle of 10 cells showed activity with a delay of about 10 degrees.

Example 6 A lyophilized supernatant obtained in the same manner as in Example 5 using human serum 2e was treated with diethylaminoethyl (DEA).
E) Adsorbed on cellulose (manufactured by Whatman), with 0.
Elute with a PH ion concentration gradient from PH 8.0 to PH 5.0 with 1M saline solution, divide the 4th fraction into 6 minutes, dialyze, desalt, and freeze-dry using polyacrylamide gel. The active ingredient was fractionated as a substance by electrophoresis, dialyzed, and lyophilized to yield 184% af cancer cell proliferation inhibitory factor C [T, D, F,)1. C) was obtained.

As shown in Example 5, this product exhibited activity of inhibiting Leukemia L 1210 cell growth curve angle in tissue culture. Ta.

Example 7 The human serum-derived cancer cell proliferation inhibitory factor A 500M&Cf/Me obtained in Examples 1 and 2 was used to examine the inhibitory effect on malignant tumor cell proliferation in tissue culture.

a I'r Mi'r L -5178Y 32.6% compared to 1ltLl.

It showed a growth-inhibiting effect of 21.3% on Sarcoma S-180 cells and 32.3% on Ehritzch's lucinoma cells, but had no growth-inhibiting effect on primary tissue culture cells of rat liver. Didn't show it.

In addition, the various malignant tumor cells tested were all shown to be viable in staining tests.

Example 8 Animal experiments were conducted using the human serum-derived cancer cell proliferation inhibitory factors A (T, D, F, H, A) obtained in Example 1.

A test animal group consisting of 5 BDF mice (2 males, body weight 20.0 f) was used.

In the first group, cancer cell growth inhibitory factor A (T, D, F.

H, A) 0.1 sy/mouse was intraperitoneally injected 24 hours after 1×10 6 Leukemia L 1210 cells were intraperitoneally injected.

In the second and third groups, the same experiment as in the first group was conducted, but the sample administration lid was 0.011 qI/mouse in the second group, and 0°001 Mv/mouse in the third group. The controls died in an average of 9.7 days, whereas the first group died in an average of 20.0 days, the second group died in an average of 15.7 days, and the third group died in an average of 12.3 days. Died in a day.

From this, the T/C value for the first group is 207. 2nd group 16
2. Group 3 showed 128.

Example 9 In a test using 500 whg/wl of human serum-derived cancer cell growth inhibitory factor B (T, D, F, H, Bl) obtained in Example 3, Leukemia L 517 in tissue culture was tested.
Approximately 15 degrees for 8Y cells, approximately 14 degrees for Sarcoma S-180 cells, approximately 5 degrees for AI Ehritzli lucinoma 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.

Example 10 Cancer cell growth inhibitory factor C (T, D, F, H, C) derived from human serum 09) obtained in Example 5 and Example 6
In a test using 00meg/ml, the cell growth curve angle was delayed by 1 degree for Leukemia L 5178 Y 1111 cells in tissue culture, approximately 22 degrees for Sarcoma 5-180m cells, and 1 degree for Ehritzli lucinoma cells. It showed the activity shown.

However, for primary tissue culture derived from rat liver,
It showed no antiproliferative effect at all. 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 factors A (T, D, F) of the present invention.
, H, A).
H, A) respectively PH3.0, PH7.0 and P
Figure 5 shows the ultraviolet absorption spectrum at H10.0.
Figures 6, 7 and 8 show the infrared I! absorption spectra of cancer cell growth inhibitory factor B (T, D, F, H0B), cancer cell growth inhibitory factor B (20) 1-, Figure 9 shows the ultraviolet absorption spectra of T, I), F, n, B) at PH3,0, PH7゜0 and 1) Hlo,0. , H, C), and Figures 10, 11, and 12 show the PH3,
0, PH7.0 and PH10.0. Applicant Kurumiki Itagami Attorney: Ken Doishi, living body (1 person)

Claims (1)

[Claims] 1) A white powder with a melting point of 245 to 249°C (decomposed) 5
10% 10% Sodium Dodecyl Sulfate Electrophoresis Molecular weight is about 73,900. Easily soluble in water, insoluble in butanol, acetone, ethyl acetate, chloroform, benzene, hexane; asparagine 110.0%, threonine 5
．． 4%, serine 3.8%, glutamic acid 16.0%
, proline 4.2 arc, glycine 1.4%, alanine 4.
8%, cystine 2.6%, valine 5.1%, methionine (), 8%. Isoleucine 2.2%, Leucine 9.6%, Tyrosine 4
．． 9%, phenylalanine 5.7%, lysine 11.4
%, histidine 3.5%, and arginine 5.2%, and the ninhydrin, Anspun, Molish, and periodic acid reactions were all positive; the infrared absorption spectrum shown in Figure 1 [KBr -: The ultraviolet absorption spectrum is 279nw in PII3.0 water.
PH7, 278nB in O water, I'H10゜()
It exhibits an absorption maximum at 277 am in water. Cancer cell growth inhibitory factor A, isolated from human blood serum
Glycoprotein JiM named (T, D, F, H, A)
. 2) It is a white powder with a melting point of 221-225°C (decomposed); the molecular weight as determined by 10% sodium dodecyl sulfate electrophoresis is approximately 22,100; easily soluble in water, insoluble in acetone, ethyl 9ate, benzene, hexane, and chloroform. Aspartic acid 16.0%, Thre A 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%
, has an amino acid composition of 3.9% phenylalanine, 7.7% lysine, 1.6% histidine, and 11.6% arginine; positive for ninhydrin reaction, negative for Anthrone, Molish, and periodic acid reactions, and 5th The figure shows the infrared absorption spectrum (KBr pellet); the ultraviolet absorption spectrum is 283Bn& and 290am in water with pH 3.0,
280B7+t in PH7.0 water, P) (10,0
A protein named cancer cell growth inhibitory factor B (T. D, F, H, B.) that exhibits maximum absorption at 2778n& in water, is separated from human blood and serum, and has a malignant tumor cell growth inhibitory effect. 3) It is a white powder with a melting point of 237 to 240°C (decomposed), and its molecular weight as determined by 10% sodium dodecyl sulfate electrophoresis is approximately 14,000; it is easily soluble in water and insoluble in acetone, ethyl acetate, benzene, hegisan, and chloroform. ; 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%,
It has an amino acid composition of 1.1% histidine and 11.6% arginine, and is positive in the ninhydrin reaction and negative in the anthrone, Molish B, and periodic acid reactions; exhibits the infrared absorption spectrum (KBr pellet) shown in Figure 9; Absorption spectrum is 278 in PH3.0 water, PII7.0
It shows maximum absorption at 290ss in water, and has a pH of 10.0.
A protein named cancer cell growth inhibitory factor C (T, D, F, H, C), which has been isolated from human surface serum and has an inhibitory effect on malignant tumor cell growth, and has an absorption maximum of 277 in water.