JPS6372700A - Protein having carcinostatic activity - Google Patents

Abstract

NEW MATERIAL:A protein separated from body fluid of larva of an insect belonging to Polyphaga suborder, having a molecular weight of about 42,000 and an isoelectric point (pI) of 8.9-9.0, resistant to trypsin and having carcinostatic activity. USE:A chemotherapeutic agent for cancer having direct cytotoxicity to cancer cell. PREPARATION:A semitransparent viscous liquid is extracted by thrusting an injection needle to the dorsal vessel of larva of Allomyrina dichotoma (a beetle). The liquid is centrifuged to remove hematocyte, heated, dialyzed with water using a cellulose dialysis membrane and subjected to salting-out with an aqueous solution of ammonium sulfate having a concentration of >=40% saturation to precipitate >=90% of protein. The precipitate is separated by filtration and treated by ion-exchange chromatography. The unadsorged fraction is subjected to reversed phase liquid chromatography to obtain the objective purified protein.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to proteins having anticancer activity.

[Problems to be solved by the conventional technology and the invention] Conventional cancer chemotherapy agents kill cancer cells by inhibiting the nucleic acid protein synthesis system, as seen in so-called alkylating agents and antimetabolites. It is. However, these drugs have problems in distinguishing between cancer cells and normal cells, and have the drawback of inevitably causing serious side effects. In addition, research is underway to isolate anticancer substances produced from animal carcinomas, but the anticancer activity of such substances is not sufficient, and the problem of side effects remains unresolved. remains as an issue.

[Means for solving problems]

Regarding animal biological defense mechanisms, research has traditionally centered on the immune system of mammals, but the present inventors have been conducting research to elucidate the biological defense mechanisms of insects.
It has been discovered that the body fluid of insect larvae belonging to the suborder Polyphagus contains a substance that exhibits strong cytotoxicity to cancer cells, and after further investigation, the present invention has been completed.

That is, the gist of the undiscovered invention lies in a protein with anticancer activity obtained from the hemolymph of an insect larva belonging to the suborder Polyphagia.

It is already known that insects have biological defense mechanisms that are different from the immune system of mammals; for example, flies and silkworms are known to produce protective substances against bacterial infections ( Gregoire, C.
, Phyeiolo Fryof engineering n5ecta.

/24c, vol. J, p. 309, Academic Press).

However, the substance according to the invention does not exhibit antibacterial action;
It is clearly different from conventionally known insect-derived physiologically active substances in that it has direct cytotoxicity against cancerous H cells. Since the substance according to the present invention is produced by the normal cells of insects and worms, it can eliminate cancer cells or their progenitor cells that may constantly appear in the animal body by exhibiting selective toxicity toward cancer cells. It should be understood as one of seven biological defense substances. The present invention will be explained in detail below.

The method for isolating the protein of the present invention and its properties are described below.

First, examples of the insects belonging to the polyphagous suborder in the present invention include capullar beetles, fireflies, chimney beetles, scarab beetles 1, stag beetles, and cicada beetles.

Which of these insects to use is determined by the ease of reproduction of larvae, the strength of body fluid obtained, and/or the strength of anticancer activity, but any insect may be used.

Insect larvae generally grow by repeatedly moulting, and insect larvae at any stage can be used. A method for obtaining body fluid from larvae includes, for example, a method of puncturing the dorsal vessel with a syringe needle to obtain body fluid without killing the larvae, and it is also possible to rebreed the larvae and obtain body fluid frequently.

It is also possible to obtain the body fluid by cutting the abdomen or by homogenizing the entire larva, but considering the subsequent purification process, it is better to extract only the body fluid purely than the method using homogenate. is desirable.

The obtained body fluid is a translucent viscous liquid, and blood cells are usually removed by centrifugation before use. In addition, since it may adversely affect the subsequent purification process, dialysis using an appropriate dialysis membrane to lower the viscosity, or heating to deactivate unnecessary hydrolytic enzymes and/or oxidative enzymes is recommended. It is preferable to add an appropriate enzyme inhibitor before treatment. In this case, it is preferable that the heat treatment is carried out at 0°C for 30 minutes under conditions such that the anticancer active protein of the present invention is not inactivated, that is, within the pH range of -9; It is preferable to heat around 7.

Next, the purification process will be explained.

The anticancer protein according to the present invention can be partially purified from body fluids by ammonium sulfate salting out method.

If the ammonium sulfate concentration at this time is 1itio% or more of saturation, the active protein will be salted out by 9Q% or more. It is possible to use not only ammonium sulfate but also sodium sulfate or organic solvents such as ethanol and acetone that have a protein salting-out effect.

It is known that body fluids contain galactose-specific lectins (Umezu et al., BiOch
em, Engineering International, / Volume 0, 5
Ti-ta page.

/9? j), it is preferable to remove these unnecessary items first. For example, DEAE-”
A method of treating body fluids with an ion exchanger such as Sephadex (5epnadsx) has been developed, and the non-adsorbed portion can be used for subsequent purification.

To further purify the anticancer active protein according to the present invention from body fluids, after the above treatment, it is further treated with an ion exchanger, and the non-adsorbed fraction is subjected to reversed phase liquid chromatography, using L-929 cells. 'fc cytotoxic activity assay.

Furthermore, an affinity chromatography method using antibodies against the two anticancer active proteins of the present invention can also be used as a purification method.

By combining the general protein purification techniques as described above, the anticancer active protein of the present invention can be isolated and produced. Seven examples of such combinations include the following purification processes.

The protein according to the present invention obtained by such a process has the following properties.

/) Molecule A is ≠ recognized, 0θ0.

There are two equal points: pI and 9 to 9,0't1''.

3) Resistant to trypsin.

4L) Second salting out with 4Lo saturated ammonium sulfate.

j) Not adsorbed to insoluble carriers containing galactose residues.

d) It is not deactivated even when heated at 0°C for 30 minutes at pHj-9.

7) Not dialyzed with cellulose membrane.

It is insoluble in organic solvents such as ethanol and acetone.

9) It has strong cytotoxic activity against cancer cells and has no antibacterial effect.

〔Example〕

Hereinafter, the present invention will be specifically explained with reference to Examples.
The present invention is not limited to the following examples unless it exceeds the gist thereof.

The anticancer activity of the present invention was determined by the following method.

RP of Mouse J-774L Cancer Cells or I, -929 Cells) iNe It4Lo medium medium size and y were placed in a 26-well microplate so that the ratio was 10'/wθ11. Add the specimen to this and incubate in a Co incubator at 37°C for 3 days.The minimum lethal concentration (L [C) And so.

Example/〈Rejuvenation〉-(1) Allomyrina dicoto-7 (Allomy
rinadichotoza L, ) (captozoan)
of larvae (weighing about 30?):! +i- A translucent viscous liquid was obtained by puncturing the vascular site with a syringe needle (approximately 2rnl/mouse). After centrifuging this liquid at J000 rpm for 30 minutes to remove collapsed spheres, it was incubated at 60°C and 75°C. The mixture was heated for 1 minute, and then dialyzed against tap water using a cellulose dialysis membrane for a whole day and night before being used in subsequent experiments (hereinafter referred to as body fluid).

The minimum lethal concentration (MI()) for J-77 Hirogan cells of larval hemolymph obtained in the first heather and winter is the dilution factor of /×7O5, and the concentration of the active substance varies depending on seasonal fluctuations in the material source and fluctuations in the growth process. No difference was observed.

(2) After heating the body fluid at 30°C for 30 minutes at the pT shown in the table below, the lethal activity against J-77≠cancer cells was measured.

Only p active substances! Even if it is heated for 30 minutes at 60°C in the range of 100 to 100°C, it will not lose its activity. When heated at 3100°C for 30 minutes, the activity completely disappeared in the pH range of 2 to 100°C.

(3) Ammonium sulfate was added to the body fluid to bring it to the temperature shown in Table 2 below, and the precipitate was collected by centrifugation, further dialyzed against distilled water, and returned to its original volume. 29 cancer cell killing activity was measured.

The active substance was salted out at a concentration above SO% saturated ammonium sulfate.

Table/ Effect of pFl on cancer cell endorsement activity when heated at 60°C for 30 minutes Table 1 Salting out of the active ingredient by ammonium sulfate pressure (4) Ion exchange the above body fluids (I) KA! -1 Sephadex'1so) processing (θ, 02 MTris-
HCt, pH7.4! ) L, and the non-adsorbed fraction was subjected to reversed phase liquid chromatography (RP-HPLO).

When the product was separated and purified using Q/column), a diffraction pattern as shown in Fig./ was obtained. When both fractions having the respective peaks in Fig. 7 are fractionated and assayed using --929, the peak of cytotoxic activity is Fr, 2 (/7.7
/7M).

<Physical properties> (1) Molecular weight After separation by reversed phase liquid chromatography (RP-HPLC!), fractions with active peaks are separated, and Laemmli (L
aemmli) system 8DE3 gel filtration (5DS-P
AGIn), a 1-molecular-weight H2KI/C band appeared.

Furthermore, this band was excised, the gel was sliced into 1/4 m squares, and after incubating overnight at μ°C in 0.7% bovine serum albumin (BSA)/phosphate buffered saline (PBS), the protein was cut into gels. Extraction, L-? When assayed by Cota, the activity was clearly observed, and the molecular weight of the active protein was found to be ≠2.

(2) Isoelectric point D E A E non-adsorbed fraction was
Two-dimensional electrophoresis was performed by the method of Rell).

From the results of 5DS-PAGE, it has been determined that a protein with a molecular weight of 42 is an active protein, so the molecule is 1.
) When the isoelectric point of the protein spot at 44.2 K was determined, it was found to be 9 to 9.0.

(3) Confirmation that it is protein 6M urea and 0. / DEIK non-adsorbed fraction 2 in SDS
Lysilen endopeptidase (Lysilen
do peptiaase, L B p ) was added at a ratio of / (molar ratio), and the mixture was digested overnight at 37°C.

After digestion, analysis was performed by SDS gel filtration (Sn2-PAGK) and liquid chromatography (HPLO), and no bands were detected in 8DS-PAGE because they were all reduced to low molecular weight. In addition, I(PLC!) showed epidemic decomposition patterns as shown in Figures D and J.

Next, when this digest was subjected to assay using I.-929, the activity was lost.

As mentioned above, this cytotoxic active factor is degraded by LEP,
It was confirmed that it was a protein by inactivation.

(4) The trypsin-digested DKAE-unadsorbed fraction was treated with tosylphenylalanylchloromethylketone (Tpcx)-)lissine at 37°C.
When the reaction was carried out overnight and analyzed by 8D8-PAGE, the active band II2 was hardly resolved. ``When assayed with fcL-929, activity was retained.

From the above, it was found that this cytotoxic active factor is resistant to trypsin.

(5) Amino acid sequence The active fraction separated by RP-HPLC was further subjected to the same O/R
After chromatography was performed again on P-H'PLO, a gas phase protein sequencer (Applied Biosys
etem Protein 8equencer4c7
When the N-terminal amino acid sequence was analyzed by
It turned out to be as shown in Figure λ.

〔Effect of the invention〕

The protein according to the present invention exhibits direct cytotoxicity against cancer cells.

[Brief explanation of the drawing]

Figure / is a diagram showing the elution pattern of the body fluid in Example / by RP-HPLC. Figure 7 shows the N-terminal amino acid sequence of the protein according to the present invention. Applicant: Mitsubishi Chemical Industries, Ltd. Agent: Yo Hase - 7 others Val Thr Thr Lys Ser Val
LysFigure 2 Thr Phe Lys Ser Asn Pro S
er'Pr.

Claims (1)

[Claims] (1) Obtained from the hemolymph of insect larvae belonging to the suborder Polyphagus, with a molecular weight of approximately 42,000 and an isoelectric point of pI 8.9 to 9.
．． 0, is resistant to trypsin, and has anticancer activity.