JPH09143082A - Protein originated from pieris papae crucivora - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein originated from Pieris rapae crucivora (common white butterfly), having specific properties such as cytotoxic activity on cultured human gastric cancer cell TMK-1, etc., exhibiting strong anticancer activity at a low concentration, having low toxicity to normal cell and expected to be useful as an anticancer agent. SOLUTION: This protein originated from Pieris rapae crucivora has the following properties. (A) Molecular weight: 6.7×10<4> to 4.4×10<5> , (B) temperature stability: stable at 40 deg.C and inactivated at 60 deg.C, (C) pH stability: stable at pH3-9, (D) solubility: soluble in pure water and PBS and insoluble in chloroform and ether, (E) enzymatic stability: stable to Pronae, Proteinase-K, Nuclease P1, (Spleen) phosphodiesterase and BAP, (F) adsorbability: adsorbable to DEAE and hydroxyapatite and free from absorptivity to phosphocellulose and Affigel Blue, (G) precipitation property: forming a precipitate with an aqueous solution of ammonium sulfate of >=40% concentration and (H) cytotoxicity: exhibiting cytotoxic activity on cultured human gastric cancer cell TMK-1.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel protein derived from the cabbage butterfly. More specifically, the present invention relates to a novel protein from the cabbage butterfly that exhibits cytotoxic activity against cancer cells.

[0002]

2. Description of the Related Art Research on useful substances in the body fluids of insects
In recent years, attention has been paid to biotechnology in connection with biotechnology, and antibacterial proteins such as cecropins and atacins and deafensins have been discovered in the study of their biodefense mechanisms, but they are effective as antitumor substances via immunity. Show Sarc
Only ophaga lectin has been reported, and no substance that directly acts on cancer cells has been discovered. Further, research and development of anticancer agents have not yet achieved their objectives sufficiently, and the emergence of more useful anticancer agents is desired.

[0003]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel anticancer agent.

[0004]

[Means for Solving the Problems] In order to search for a substance having an anticancer activity in insects, the present inventors have a cytotoxic activity against human gastric cancer cultured cells TMK-1 (hereinafter referred to as TMK-1). By screening lepidopteran body fluids using as an index, the cabbage butterfly ( Pierisrapae cr
ucivora) has a strong cytotoxic activity, and the present invention has been completed based on this finding. The present invention
This is a factor derived from the cabbage butterfly, which is contained in body fluids of larvae, pupae, or adults and has a strong cytotoxic activity against cancer cells. Furthermore, the present inventors have confirmed that the white-spotted butterfly, the black-spotted butterfly and the white-banded white butterfly also contain the same factor.

Then, it was confirmed that this substance has the following properties using the cytotoxic activity against TMK-1 as an index. (A) Molecular weight: 6.7 × 10 ^{4 to} 4.4 × 10 ⁵ (b) Stable at 40 ° C., deactivated at 60 ° C. (c) Stable at pH 3.0 to 9.0 (d) Pure water, PBS Dissolved in chloroform and insoluble in chloroform and ether (e) Pronase, Proteinase-K, N
sequence-P1, Phosphodiester
ase, Spleen Phosphodiester
Stable to ase or BAP (f) DEAE or Hydroxyapatite
Adsorbed by Phosphocellulose or Affigel blue (g) A precipitate is formed in 40% or more ammonium sulfate aqueous solution (h) It does not show cytotoxicity to erythrocytes and lymphocytes. Administration methods such as intramuscular injection, intramuscular injection, and intravenous injection are possible, but intravenous injection is preferable. The dose varies depending on the individual difference of the patient, the condition of the disease, etc.
g. Acute toxicity is intravenous toxicity to mice LD ₅₀
The value was 2.2 mg / kg, which was confirmed to be less toxic than existing anticancer agents.

[0006]

[Details of action] Although details are not clear, observation of how TMK-1 to which the protein of the present invention derived from the cabbage butterfly was administered undergoes cell injury is similar to that when cells express apoptosis and cause cell death. , It may be involved in the expression of apoptosis in cancer cells.

[0007]

【Example】

1) Preparation of worm body extract After collecting the worm body, it was stored in a -50 ° C freezer and used for the experiment. Extraction was carried out by adding 10 times the amount of PBS of the worm and compression extraction,
After centrifugation at 5000 rpm for 15 minutes, the supernatant was filtered through a 0.22 μm filter to remove insoluble precipitates, which was stored as a 10-fold diluted body sample in a -50 ° C. freezer for use. 2) Detection of cytotoxicity by parasite extract TMK-1 cells, which are cultured human gastric cancer cells, were used, and the medium was RDF (10% FBS). The sample was diluted 10-fold with the culture medium, and trypsin-ED was added to this.
TMK-1 cells by TA treatment were inoculated with 10 ⁴ / ml, 37
Culturing was performed at 48 ° C. for 48 hours. To determine the cytotoxic activity, plate detachment due to cell destruction and life-and-death determination by trypan blue staining were performed. When almost half of the cells were killed, the cytotoxic activity was determined to be 1000-fold or more. Those with a 100-fold dilution or less were determined to be negative. By this method, cytotoxic activity in various parasite extracts of butterflies was examined.

3) Change in cytotoxicity due to concentration After diluting the sample in 96-well plate with 10 ⁴ / ml TMK-1 cells as 100 μl / well at 37 ° C. for 48 hours, the viable cells were subjected to the KitII (XTT) method. Was developed by and measured by ELISA. 4) Change in cytotoxic activity by heat treatment 100 μl of 100-fold diluted sample placed in an Eppendorf tube in each water bath at 40 ° C, 60 ° C, 80 ° C, 100 ° C was heated for 1 hour to activate the activity by the method of 2). I checked. 5) Changes in cytotoxic activity due to pH treatment pH 1.0, 3.0, 5.0, 7.0, 9.0, 11.0, 1
The sample was placed in a tube so that the concentration was 100 times diluted in 100 μl of each buffer of 4.0, and the activity was examined by the method of 2) after 1 hour at 37 ° C. 6) Change of cytotoxic activity by enzyme treatment and chemical identification Pronase 1 mg / ml (0.01 M Tris, pH 7.8), Proteinase-K 50
ug / ml (0.01M Tris, pH7.8), Nuclease-P1 5mg / ml (10 / N
buffer), Phosphodiesterase 4U / μl (7 / PDase buffe
r), Spleen Phosphodiesterase 0.1 U / μl (7 / PDase buf
fer) and BAP (10x), add a body fluid sample to make the final concentration of the body fluid sample 100-fold, shake gently, then heat at 37 ° C for 1 hour in a water bath to activate the activity. I checked.

7) Adsorption experiment by column DEAE (10mM Tris.CL, pH7.5), Phosphocellulose (10mM
HO PO ₄ , pH 7.5), Affigel blue CM (10 mM Tris.Cl, pH 7.
5), P inside the processing column (1 mm inner diameter x 2 cm length glass tube) packed with Hydroxyapatite (10 mM NaPO ₄ , pH 7.0)
Wash with 100 ml of BS (4 ml) and 100-fold diluted sample
After adding μl and shaking for 1 hour, centrifuge, remove the supernatant (“supernatant” in Table 4), wash the precipitate twice with 4 ml of PBS (“wash solution” in Table 4), and eluate (10 mM Tris.Cl). -1M NaCl,
100 μl of pH 7.5) was added, and the mixture was shaken for 1 hour and then centrifuged to obtain a supernatant (“eluate” in Table 4), and each activity was examined by the method of 2). 8) Precipitation experiment with ammonium salt Saturation of ammonium sulfate, 80%, 60%, 50%, 4
10 mL of the sample was added to 1 ml of 0%, 20%, or 10% aqueous solution.
The mixture was added so as to be diluted 0-fold, and after 24 hours at 5 ° C., the centrifugal precipitate was washed twice with ammonium sulfate having the same concentration, the precipitate was dissolved in 1 ml of PBS, and the activity was examined by the method of 2).

9) Elution experiment with organic solvent 100 μl each of chloroform and ether was mixed with 100 μl of a 100-fold diluted sample, the aqueous layer was removed, the solvent was evaporated under vacuum, and the residue was dissolved in 100 μl of PBS. ). 10) Measurement of molecular weight by molecular filtration Centricon 3Kd, 10Kd, 30Kd, 50Kd, 100Kd
In order to examine the passage of a substance having a cytotoxic activity against TMK-1 contained in 1 ml of a 100-fold diluted sample passed through centrifugation at 3000 rpm, the activity of each of the passing liquid and the non-passing substance was examined by the method of 2). did. The non-passing substance was dissolved in 1.0 ml of PBS and used for the measurement. 11) Measurement of molecular weight by gel filtration 10-fold diluted sample was filtered with a filter (0.22 μl) to remove insoluble matter, and Sumarart SMRART system was used. The column was SUPEROSE 6PC 3.2 / 30 and the eluent was 20 mM phosphate buffer. (PH7.2) + 0.3M NaCl, flow rate: 40μl / mi
n, detection: UV 260 was performed, and the activity of each fraction eluate was examined by the method 2).

2. Results A) Cytotoxic activity in various butterflies 1) By the method of 2), 13 of swallowtail butterfly, black swallowtail butterfly, crow swallowtail butterfly, lycaenoptera swallowtail butterfly, stag beetle, pierced butterfly, Okinawa butterfly, zebra butterfly, butterfly butterflies, butterfly, omurasaki, kabata butterfly, silkworm Seeds of pupae and Pieris rapae, Colias, Cabbage butterfly, Lycaenidae,
As a result of investigating 6 kinds of adults of Pleurotus cornucopiae and Bombyx mori, cytotoxic activity was observed in pupa of cabbage butterflies and adult body fluids diluted 10,000 times or more, but not in others. B) Cytotoxic activity in cabbage butterfly Dose response of cytotoxic activity in cabbage butterfly body fluid by the method of 3) is shown in Fig. 1, and the cytotoxic activity of pupa (1, 2) is 1/1000, 000,
1 / 100,000 for larvae and 1 / 10,000 for adults T
It shows a strong activity (> IC ₅₀ ) against MK-1 cultured cells. C) Change in cytotoxic activity by heat treatment The activity is retained at 40 ° C or lower, but 60 ° C, 80
It is deactivated at 100 and 100 ° C (Table 1). In the following tables, "-" indicates negative and "+" indicates positive, which is almost IC ₅₀ . Moreover, the cytotoxic activity increases as the number of + increases.

(Table 1) Temperature Sample dilution rate 40 ° C 60 ° C 80 ° C 100 ° C Untreated 1/1000 +++ − − − +++ 1/10000 +++ − − − +++

D) Change in cytotoxic activity by pH treatment The activity is retained at pH 3.0, 5.0, 7.0 and 9.0, but deactivated at pH 1.0 or lower or 11.0 or higher. Yes (Table 2).

(Table 2) pH sample dilution rate 1.0 3.0 5.0 7.0 9.0 11.0 14.0 untreated 1/1000 − +++ +++ +++ +++ − − +++ 1/10000 − +++ ++ + +++ +++ − − +++

E) Change of cytotoxic activity by enzyme treatment Protease (Pronase, Proteinas)
e-K), nuclease (Nuclease-P1), esterase (Phosphodiesterase, Spleen Phosphodiesterase) and BAP all retain their activity. It is considered that the protease does not act due to the inhibition of the action of lipids and the like bound to the active substance (Table 3).

[0016] (Table 3) enzyme sample dilution rate Protease Proteinase-K Nuclease-P1 Phospho BAP diesterase 1/1000 +++ +++ +++ +++ +++ 1/10000 +++ +++ + ++ +++ +++

F) Adsorption of cytotoxic substance by column Adsorbed on DEAE and Hydroxyapatite and well eluted by the eluent, Phosphocellulose and Affigel
It is not adsorbed by blueCM (Table 4).

(Table 4) Column DEAE Phosphocellulose AffigelBlueCM Hydroxiapatite Sample dilution rate 1/1000 1/10000 1/1000 1/10000 1/1000 1/10000 1/1000 1/10000 Untreated +++ +++ +++ +++ +++ +++ +++ +++ Supernatant − − +++ +++ +++ +++ − − Wash solution − − +++ +++ +++ +++ − − Eluate +++ +++ ＋ － ＋ － +++ +++

G) Precipitation of Cytotoxic Substances in Ammonium Salt Solution Precipitated with 50% or more of ammonium sulfate, partially precipitated with 40%, and not precipitated with 20%. H) Elution of cytotoxic factor with organic solvent No elution with chloroform or ether was observed. Further, phenol was highly toxic to cells and could not be determined. I) Measurement of the molecular weight of the cytotoxic active substance.
Since the passage of the active factor at d is not observed and it does not pass below 100 Kd of the centricon, the active substance is 1
It was considered to be slightly larger than 00 Kd (Table 5).

(Table 5) Sample centricon dilution ratio 3Kd 10Kd 30Kd 50Kd 100Kd Passed liquid 1/1000 − − − − − 1/10000 − − − − − Impenetrable substance 1/1000 +++ +++ +++ + ++ +++ 1/10000 +++ +++ +++ +++ +++

Next, the molecular weight of the cytotoxic activator was measured by gel filtration. As a result, the fractions 13 to 14 had peaks, and it is considered that the molecular weight is about 1.5 × 10 ⁵ on the graph. The activity is 6.7 × 10 ^{4 to} 4.4.
It is in the portion of × 10 ⁵ . When calculated from the retention time, it is considered to be about 1.5 × 10 ⁵ .

[0022]

INDUSTRIAL APPLICABILITY The protein derived from the cabbage butterfly of the present invention exhibits strong anticancer activity at a low concentration. Further, since it has low toxicity to healthy cells, it is expected to be used as an anticancer agent.

[Brief description of the drawings]

Figure 1: Measurement of viable cells by ELISA

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Keiji Wakabayashi 2-5-28 Higashigaoka, Meguro-ku, Tokyo (72) Inventor Kazunori Koiwai 1500 Inoguchi, Nakai-cho, Ashigarakami-gun, Kanagawa Terumo Corporation

Claims (1)

[Claims] 1. A protein derived from the Pieris rapae crucivora derived from the Pieris rapae crucivora having the following properties (a) to (h): (a) molecular weight: 6.7 × 10 ^{4 to} 4.4 × 10 ⁵ (b) at 40 ° C. Stable, deactivated at 60 ° C (c) Stable at pH 3.0 to 9.0 (d) Soluble in pure water and PBS, insoluble in chloroform and ether (e) Pronase, Proteinase-K, N
sequence-P1, Phosphodiester
ase, Spleen Phosphodiester
Stable to ase or BAP (f) DEAE or Hydroxyapatite
Adsorbed on Phosphocellulose or Affigel blue (g) A precipitate is formed in 40% or more ammonium sulfate aqueous solution (h) Cytotoxic activity against human gastric cancer cell line TMK-1