JP2014002043A - New screening method for anti-infiltration drug, and anti-infiltration agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for rapidly and efficiently screening for an anti-infiltration agent, and the anti-infiltration agent obtained by the method.SOLUTION: There are provided a method for identifying a substance having an anti-infiltration action, and an anti-infiltration agent obtained by the method. The method includes the steps of: (a) providing each of cytoplasm suspension liquid containing labeled actin, and containing or not containing a candidate substance; (b) adding liposome which contains phospholipid having negative charge to the cytoplasm suspension liquid, and stimulating actin fiber formation; (c) measuring actin fiber formation degrees; (d) obtaining, as an actin fiber formation rate, a ratio of the actin fiber formation degree under the presence of the candidate substance to the actin fiber formation degree under the absence of the candidate substance; and (e) evaluating the anti-infiltration action of the candidate substance by using an actin fiber formation rate value as an indicator.

Description

  The present invention relates to a method for screening a substance having an anti-invasive action in the fields of cancer therapy and anti-cancer drug development, and an anti-invasive agent obtained by the screening method.

  When cancer cells invade and metastasize into normal tissues, the cells form a raffle membrane in the direction of progression, and while the cell membrane progresses, they form filamentous and lamellipodia, and use it as a scaffold It is known to move.

  In the past, as a method for predicting the risk of cancer invasion and / or metastasis, identification of a specific nucleic acid or polypeptide marker has been actively attempted, for example, as a marker for predicting oral cancer invasion. A gene that exhibits high expression in cells with high invasive ability has been studied (Patent Document 1). This diagnostic marker isolates cells with high invasive ability from cultured cell lines established from oral cancer patients using an in vitro invasion assay, and is a gene showing high expression in cells with high invasive ability, IFITM1 and WNT5B. It is obtained by identification.

  Various compounds have already been reported as anti-invasive agents. For example, a 3-cyano-quinoline derivative is described as a non-receptor tyrosine kinase inhibitor, a low molecular compound as a dynamin function inhibitor, or a quinazoline derivative as a MAPK kinase pathway inhibitor or non-receptor tyrosine kinase inhibitor. (Patent Documents 2 to 4).

  On the other hand, the above-mentioned raffle film formation and pseudopodia formation are backed by a cytoskeleton centered on fibers in which a protein called actin is polymerized. That is, it is also known that actin fibril formation is one of the most important factors during cell infiltration (Patent Document 5).

  However, since there are many proteins and factors involved in the formation of these actin fibrils, actin polymerization is often not efficiently inhibited even when inhibitors of each protein are used, and drugs that suppress actin polymerization are directly screened. To do so far has not been reported.

  On the other hand, the present inventors have succeeded in establishing a certain method for evaluating the formation of actin fibrils in vitro and the degree of formation thereof. By this system, actin, a known dynamin inhibitor and actin inhibitor Participation in polymerization has been confirmed (Non-Patent Documents 1 and 2).

JP 2008-206482 A JP 2006-508944 A Japanese Patent Laid-Open No. 2004-51895 Japanese Patent Laid-Open No. 2003-579751 JP 2009-57364 A

Biochemical and Biophysical Research Communications, 2009, Vol.390, No.4, pp.1142-1148. The Journal of Biological Chemistry, 2009, Vol.284, No.49, pp.34244-34256.

  An object of the present invention is to provide a screening method capable of quickly and efficiently identifying a substance having an anti-invasive action from candidate substances, and an anti-invasive agent obtained by the screening method.

  The present inventors have established a technique for forming actin fibrils in vitro. Surprisingly, by using the actin formation rate quantified using this technique, the candidate substances can be selected. From these results, it was found that a substance having an anti-invasive action could be screened, and the present invention was completed. That is, the actin fiber formation rate obtained by the method of the present invention shows an excellent correlation with the anti-invasion action in cells. In general, since actin fibril formation in a living body is based on a mechanism involving many factors as described above, anti-invasion of a large number of candidate substances can be achieved by using the actin fibril formation inhibitory action in such a simple system in vitro as an index. It was very surprising that the action could be evaluated quickly.

That is, the present invention is a method for identifying a substance having an anti-invasive action,
(A) providing each cytoplasmic suspension comprising labeled actin and with or without a candidate substance;
(B) adding a liposome containing a negatively charged phospholipid to the cytoplasmic suspension to stimulate actin fibril formation;
(C) measuring the degree of actin fibril formation,
(D) obtaining a ratio of the degree of actin fiber formation (B) in the presence of the candidate substance to the degree of actin fiber formation (A) in the absence of the candidate substance as an actin fiber formation rate, and (e) actin fiber formation The present invention relates to a method including a step of evaluating an anti-invasive action of a candidate substance using a rate value as an index.

  In the method of the present invention, it is preferable to further include ATP and an ATP regeneration system in the step (a).

  In the method of the present invention, the labeled actin is preferably pyrene actin.

  In the method of the present invention, the cytoplasmic suspension is preferably prepared from brain cells, hepatocytes or testis cells.

  In the method of the present invention, the anti-invasive action is preferably a raffle film formation inhibitory action, a cell migration inhibitory action and / or a cell infiltration inhibitory action.

  The present invention also relates to an anti-invasive agent comprising N '-(4- (dipropylamino) benzylidene) -2-hydroxybenzohydrazide.

  In a preferred embodiment of the present invention, the anti-invasive agent is an antitumor agent, a cancer cell migration inhibitor, a cancer cell infiltration inhibitor, an anti-metastatic agent, or an immunosuppressant.

  According to the method of the present invention, the anti-invasive action of a large number of candidate substances can be screened quickly and effectively, and for example, high throughput can be achieved with a plate reader. In fact, the present inventors screened 150 or more compounds randomly extracted from a molecular library, and confirmed the effectiveness of the screening method of the present invention.

  N ′-(4- (dipropylamino) benzylidene) -2-hydroxybenzohydrazide discovered by the screening method of the present invention effectively inhibits the formation of a raffle film and also inhibits the migration of cancer cells. It has been confirmed to show invasive action, and can be used as a pharmaceutical composition, for example, an anti-invasive agent, an antitumor agent, a cancer cell migration inhibitor, an anti-metastatic agent, or an immunosuppressant.

FIG. 4 is a schematic diagram (a) illustrating in vitro (in vitro) polymerization of actin and a schematic diagram (b) illustrating a principle of measuring actin fiber formation amount using pyrene actin. It is explanatory drawing of the calculation method of actin fiber formation rate. It is a figure which shows the structural formula of a test compound. It is a graph which shows the result of the actin fiber formation rate of a test substance. It is a fluorescence micrograph of rhodamine staining showing the effect of suppressing raffle film formation by serum stimulation. It is a microscope picture which shows the result of a wound healing assay. It is a graph which shows the result of a wound healing assay.

  In the present invention, the “anti-invasive action” means an action that suppresses an invasion process in which cancer cells enter the surrounding tissue and proliferate. When invasion occurs, cancer cells enter the blood and enter the other organs and tissues in a hematogenous manner and metastasize. For example, since the formation of actin fibrils in cells is essential for the invasion process, if actin fibril formation can be efficiently suppressed, cancer cells cannot enter other tissues, and one place Therefore, metastasis can be suppressed.

  In the present specification, the term “actin fibril formation” means that monomeric G actin and G actin polymerize to form fibrous actin (also referred to as F-actin). -Synonymous with actin formation.

  The “candidate substance” in the present invention is not particularly limited in terms of molecular weight and compound composition as long as it is soluble or dispersible in an aqueous solution. Examples include, but are not limited to, low molecular organic compounds, high molecular organic compounds, natural organic compounds, and crude drugs.

  The screening method of the present invention is a method for identifying a substance having an anti-invasive action, comprising: (a) providing a cytoplasmic suspension containing labeled actin and containing or not containing a candidate substance; b) adding liposomes containing negatively charged phospholipids to the cytoplasmic suspension to stimulate actin fibril formation, (c) measuring the degree of actin fibril formation, (d) absence of candidate substances A step of obtaining a ratio of the degree of actin fiber formation (B) in the presence of the candidate substance with respect to the degree of actin fiber formation (A) below as an actin fiber formation rate, and (e) a candidate substance using the value of the actin fiber formation rate as an index The step of evaluating the anti-invasive action of

In the screening method of the present invention, step (a) is a step of providing each cytoplasmic suspension containing a labeled actin and containing or not containing a candidate substance, but more efficiently forms actin fibers. Therefore, it is preferable to further contain ATP, GTP, ATP regeneration system (CPK (creatine phosphokinase), CP (creatine phosphate) in addition to ATP) and the like. As the cytoplasm, cytoplasms derived from various cells such as brain cells, hepatocytes, testis cells and the like can be used, but brain cytoplasm is more preferable from the viewpoint of high actin fiber formation rate. The origin of the cell is not particularly limited, but from the viewpoint of screening for anti-invasive agents, mammals used in human drug evaluation systems, such as rats, mice, guinea pigs, rabbits, pigs, monkeys, dogs, etc. are more preferable. . The buffer used for suspending cytoplasm is a buffer that does not contain inorganic phosphorus, and is used in the biochemical field, such as a good buffer adjusted to around physiological pH 7.4. If it is, it can be used without particular limitation, and examples thereof include HEPES, MES, and PIPES. KCl, MgCl ₂ , CaCl _2, etc. can be added to this buffer solution so as to be approximately isotonic and optimal in composition and concentration for actin fiber formation, and the viscosity can be adjusted with sucrose or the like. Furthermore, it is also preferable to add DTT for the purpose of activating the protein in the suspension or EGTA for the purpose of adjusting the calcium concentration. The cytoplasmic concentration of the cytoplasmic suspension is preferably about 3 to about 30 mg / ml, more preferably about 6 to about 20 mg / ml, and most preferably about 8 to about 10 as the final concentration when used in step (b). 14 mg / ml. When the cytoplasm concentration is lower than 3 mg / ml, actin fiber formation tends to be not achieved successfully, and when it is higher than 30 mg / ml, the viscosity becomes high and the actin fiber formation tends not to be accurately quantified.

  Examples of the label for the labeled actin used in the present invention include pyrene, rhodamine, fluorescein and the like, but are not limited to these as long as it enables detection of actin or actin fibers. As will be described later, it is preferable to use pyrene because formation of actin fibers can be quantitatively detected.

In the screening method of the present invention, the step (b) is a step of stimulating the formation of actin fibers in the cytoplasmic suspension prepared in the step (a) by adding a liposome containing a phospholipid having a negative charge. It is. The phospholipid having a negative charge is not particularly limited, and examples thereof include phosphatidylserine (PS), phosphatidylinositol-4,5-diphosphate (PIP ₂ ), and other phospholipids such as phosphatidylcholine ( PC) and the like to form stable liposomes.

  Specifically, the step (a) and the step (b) can be carried out, for example, by adding liposomes containing phosphatidylserine (PS) in the presence of ATP in the mouse brain cytoplasm (Non-patent Document 2).

  In the screening method of the present invention, the step (c) is a step of measuring the degree of formation of actin fibrils, but the method depends on the type of labeled actin and is spectroscopically or before and after stimulation of actin fibril formation. Methods such as assessing morphological changes can be used.

  Specifically, when actin is labeled using pyrene having the characteristic that the intensity of a specific wavelength increases as the density increases, the actin fibril formation amount is determined based on the specific wavelength of this pyrene (excitation wavelength: 365 nm, fluorescence wavelength: 407 nm). Can be measured spectroscopically. This method has been established and is preferable because it can be quantitatively measured with high accuracy because the degree of actin fibril formation and the amount of fluorescence are well correlated. This method is not limited to pyrene, and a label having the same characteristics as pyrene can also be used.

  For example, as one embodiment of the present invention, the measurement of the degree of actin fibril formation using pyrene is a phospholipid having a negative charge in a brain cytoplasm suspension containing pyrene actin and ATP, to which only a candidate substance or a solvent is added. Is added, and the fluorescence intensity is measured over time with a fluorescence spectrophotometer until the change in fluorescence reaches equilibrium (approximately 1000 to 2000 seconds) after the addition.

  In addition, when using actin labeled with a molecule whose fluorescence can be detected by a fluorescence microscope such as rhodamine or fluorescein as the labeled actin, in the screening method of the present invention, after inducing actin fibril formation, under a fluorescence microscope, The actin fibril formation degree can be evaluated morphologically by observing the actually formed actin fibrils directly with a fluorescence microscope, acquiring the microscope images with a camera, and comparing them.

  The actin fibril formation rate required in the step (d) of the screening method of the present invention is the degree of actin fibril formation (A) in a control actin reproduction system that does not include a candidate substance, and is 100 in the presence of a candidate substance. It shows the ratio of the degree of actin fiber formation (B) (FIG. 2).

  In the step (e) of the screening method of the present invention, when the anti-invasive action is evaluated using the value of actin fibril formation rate as an index, the actin fiber formation rate is decreased from 100, preferably about 70 or less. More preferably, the test substance that is about 30 or less has an anti-invasive effect.

  N ′-(4- (dipropylamino) benzylidene) -2 is a compound in which an actin fibril formation inhibitory action and an anti-invasive action have been identified by the screening method of the present invention and a high anti-invasive action has been confirmed in cell assays. -Hydroxybenzohydrazide (also referred to herein as Compound No. 1). N '-(4- (dipropylamino) benzylidene) -2-hydroxybenzohydrazide is commercially available (Vitas-M Laboratories) or can be produced by a conventional method.

  Therefore, according to the present invention, there is provided an anti-invasive agent comprising N '-(4- (dipropylamino) benzylidene) -2-hydroxybenzohydrazide or a pharmaceutically acceptable salt thereof.

  Similar compounds of N ′-(4- (dipropylamino) benzylidene) -2-hydroxybenzohydrazide have recently been reported as inhibitors of AMP-activated protein kinase (AMPK) (International Publication No. 1). 2012/0275548) is not known at all for its involvement in actin fibril formation.

  The anti-invasive agent of the present invention is effective for cell activities in which actin fibril formation is essential, such as macrophage phagocytosis, cancer cell migration, and metastasis. Therefore, the anti-invasive agent of the present invention can be used as an antitumor agent, a cancer cell migration inhibitor, an anti-metastatic agent, an immunosuppressant, and the like.

  Examples of pharmaceutically acceptable salts include basic addition salts, acid addition salts, and the like. Examples of basic addition salts include alkali metal salts such as sodium salts and potassium salts; for example, calcium salts and magnesium salts. Alkaline earth metal salts; for example ammonium salts; organic amine salts such as trimethylamine salt, triethylamine salt, dicyclohexylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, procaine salt and N, N′-dibenzylethylenediamine salt Examples of acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate and perchlorate; for example, acetate, formate, maleate, fumarate, and tartaric acid. Salt, citrate, ascorbate and trifluoroacetate Organic acid salts of; for example methanesulfonate, isethionate, and sulfonates such as benzenesulfonate and p- toluenesulphonate salts.

  Examples of the administration method of the anti-invasive agent of the present invention include intravenous, arterial, oral, intramuscular, subcutaneous, intraperitoneal, nasal drop, eye drop, ear drop, transdermal, and local administration.

  Examples of the dosage form of the anti-invasive agent of the present invention include tablets, powders, granules, capsules, liquid injections, nasal drops, eye drops, ear drops, suppositories, and patches.

  The dose of the anti-invasive agent of the present invention varies depending on various conditions such as age, weight, symptoms, etc., but is usually about 1 to 100 mg / kg.

  Screening is performed according to the method of the present invention, and for substances found to inhibit actin fibril formation in vitro, the anti-invasive effect can be confirmed by a conventionally used evaluation method.

  Examples of such a method include a method for evaluating inhibition of raffle film formation and inhibition of cell invasion in a wound healing assay.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Formation of actin fibers (1-a) Preparation of mouse brain cytoplasm According to Yamada et al., J. Biol. Chem., 2009, Vol.284, pp.34244-34256, 20 brains of mice (strain C57BL6J) 5 ml of XB buffer (10 mM HEPES, 100 mM KCl, 2 mM MgCl ₂ , 0.1 mM CaCl ₂ , 5 mM EGTA, 50 mM sucrose, 1 mM dithiothreitol, 1 μg / ml leupeptin, 5 μg / ml pepstatin, and 0.4 mg / ml ml fluorinated phenylmethylsulfonyl) pH 7.4. The suspension was centrifuged at 3,000 × g for 20 minutes and 10,000 × g for 20 minutes. The resulting supernatant was diluted 4-fold with XB buffer and centrifuged at 400,000 × g for 1 hour. The clear supernatant was carefully collected and concentrated to a quarter volume using a Centriprep-10 concentrator (Amicon). The final concentration of cytoplasm was 40-50 mg / ml.

(1-b) Actin fibril formation reaction 40 U / ml CPK (creatine phosphokinase), 8 mM CP (creatine phosphate) so that the mouse brain cytoplasm obtained in (1-a) has a final concentration of 12 mg / ml. Diluted in XB buffer containing 1 mM ATP, 0.286 mg / ml pyreneactin and 0.1 mM GTP, the test substance was added and mixed. After incubating for 10 minutes, actin fiber (F-actin) formation reaction was initiated by adding 5 μl of 120 μM liposome (50% PC, 50% PS: Avanti Polar Lipid) to 65 μl of the mixture. Pyrene actin was purchased from Cytoskeleton. Other reagents were purchased from Wako Pure Chemical. The test substance shown in Table 1 (FIG. 3) was dissolved in DMSO (dimethyl sulfoxide) and used as a negative control, and the same amount of DMSO as the solvent of the test substance was added. The final concentration of the test substance in the sample was 40 μM, and the dilution was adjusted so that the solvent DMSO in which the test substance was dissolved was final 1% (v / v, in the sample).

(1-c) Evaluation Method for Actin Fiber Formation Rate The amount of F-actin formation was quantified using the change in the fluorescence intensity of pyrene derived from pyrene-labeled actin as an index (FIG. 1). As the density of pyrene increases, the fluorescence intensity at 407 nm increases (excitation wavelength: 365 nm, fluorescence wavelength: 407 nm). This fluorescence intensity is known to be proportional to the amount of actin fibers formed. The change in fluorescence intensity was measured using a fluorescence spectrophotometer (F-2500HITACHI, manufactured by Hitachi), and the change with time in the amount of F-actin formed was measured. The amount of F-actin formation was determined by measuring the amount of increase in fluorescence intensity after the addition of liposomes until 2000 seconds when the fluorescence change of pyrene was in equilibrium (FIG. 2). When the test substance was not included, the increase was 100% and standardized.

  The results are shown in FIG. As a positive control, Dynasoa showed an actin fibril formation inhibitory effect with an actin fibril formation rate as high as 35.3% of the control, confirming that the evaluation system is appropriate. And compound no. 1 showed a very high actin fibril formation inhibitory effect of 28.5%. On the other hand, Compound No. 2-No. 6 was 98.5%, 95.6%, 108.8%, 104.5% and 107.8% of the control, respectively, showing no actin fibril formation inhibitory effect.

  From this result, Compound No. 1 is considered to be a candidate as an actin fibril formation inhibitor and an infiltration suppressor. 2-No. About 6, it suggests that the anti-invasive action in the cells cannot be expected.

Example 2 Examination of Raffle Film Formation Inhibition in Lung Cancer Cell Line (2-a) Culture of Lung Cancer Cell Line Human lung cancer cell line (H1299: ATCC No. CRL-5803) was obtained from 10% fetal bovine serum (FBS) (Invitrogen) The cells were cultured in a Dulbecco's modified Eagle medium (DMEM: Invitrogen) at 37 ° C. in the presence of 5% CO ₂ .

(2-b) Raffle membrane formation by serum stimulation Put a collagen-coated cover glass (Iwaki Glass Co., Ltd.) in a 6-well plate so that the human lung cancer cell line H1299 is 2 × 10 ⁴ cells / well and culture for 2 days. After that, the medium was replaced with 2 ml of DMEM containing 0.1% FBS, and further cultured for 16 to 24 hours. Thereafter, the medium was replaced with 2 ml of DMEM containing 10% FBS, and the temperature was kept for 10 minutes. The cells were quickly washed with ice-cooled phosphate buffered saline (PBS), and then fixed with PBS containing 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd.) at room temperature for 20 minutes. After fixation, the cells were permeabilized with PBS containing 0.1% TX-100 (Sigma-Aldrich Japan). Furthermore, after blocking with PBS containing 10% donkey serum, the cells were treated with rhodamine-modified phalloidin (Molecular Probes) to stain intracellular actin fibers. After the staining treatment, the slide glass was mounted using a mounting agent (Diagonistic Biosystems) according to a conventional method. When investigating the raffle film formation inhibitory effect of the test substance, 2 ml of 0.1% FBS-DMEM in which the test substance of Table 1 was dissolved to 40 μM and the cells were kept at 37 ° C., and 30 minutes before stimulation with serum Administered to the cells. Since each compound was dissolved in dimethyl sulfoxide (DMSO), the control containing no test substance was observed using the same volume of DMSO instead of the compound solution, and the acquired image was Adobe Photoshop (registered). (Trademark) CS3 (Adobe Systems Incorporated) and Adobe Illustrator (registered trademark) CS3 (Adobe Systems Incorporated) were used for editing.

  The results are shown in FIG. In cells that are not serum-stimulated, almost no raffle membrane is formed at the cell edge, but in the control, formation of a raffle membrane 1 in which F-actin is abundant as shown by the arrow is observed during serum stimulation. It was. Compound No. In the system administered with 2 to 6, the raffle film is formed in the same manner as the control, whereas compound No. In the 1 administration group, almost no raffle film was formed. 1 actin fibril formation inhibitory effect was confirmed at the cellular level.

  From this result, it can be seen that the actin fibril formation evaluation system at the test tube level in Example 1 shows a good correlation with the anti-invasive action in cancer cultured cells.

Example 3 Examination of Cell Migration Inhibition by Wound Treatment Assay (3-a) Cell Culture H1299 cells were cultured in the same manner as in Example 2.

(3-b) Wound healing assay H1299 cells were cultured to become confluent in a glass bottom dish (Iwaki Glass Co., Ltd.). One day before assay, change the culture medium to 0.1% FBS-DMEM and incubate for 16-20 hours. Thereafter, the cells were randomly scraped at four points at regular intervals using a yellow chip. One dish was set to 0 hours, fixed with methanol, and immediately stained with Giemsa (Sigma-Aldrich). Other dishes include control-free dysoa and compound no. 1 and compound no. 2, 2 ml of 0.1% FBS-DMEM in which the test substance was dissolved to 40 μM was kept warm and administered immediately after wound creation. Controls containing no test substance were observed using the same volume of DMSO instead of the compound solution. The cells observed over time were further cultured for 9 hours under the conditions of 37 ° C. and 5% CO ₂ . After culture, the cells were fixed with methanol and stained with Giemsa. The cells subjected to Giemsa staining were observed with a microscope to obtain an image. In order to evaluate how much gaps the cells filled, five areas were randomly selected in the same petri dish, and the filled areas were measured from the image using ImageJ (NIH), and the average value was calculated. .

  The results are shown in FIGS. From FIG. 6, the degree of cell migration is measured from the left and right of the part where the cell is mechanically scraped, and the infiltrating ability of the cell is illustrated with the area where the wounded part of the control fills the wound area as 100 7 Compound No. In the 2 administration group, the infiltration ability was 97.5%, which is almost the same as that of the control, but in the positive control dynasoa administration group, 53.3%, Compound No. In one administration group, the invasive ability of cancer cells was remarkably suppressed at 70.7%.

  This shows that the actin fibril formation inhibitory action at the test tube level in Example 1 was confirmed with good correlation as an infiltration suppressing effect in experiments using cultured cancer cells.

1 Raffle membrane 2 Wound part

Claims (7)

A method for identifying a substance having an anti-invasive action,
(A) providing each cytoplasmic suspension comprising labeled actin and with or without a candidate substance;
(B) adding a liposome containing a negatively charged phospholipid to the cytoplasmic suspension to stimulate actin fibril formation;
(C) measuring the degree of actin fibril formation,
(D) obtaining a ratio of the degree of actin fiber formation (B) in the presence of the candidate substance to the degree of actin fiber formation (A) in the absence of the candidate substance as an actin fiber formation rate, and (e) actin fiber formation A method comprising a step of evaluating an anti-invasive effect of a candidate substance using a rate value as an index. The method according to claim 1, wherein the step (a) further comprises ATP and an ATP regeneration system. The method according to claim 1 or 2, wherein the labeled actin is pyrene actin. The method according to any one of claims 1 to 3, wherein the cytoplasmic suspension is prepared from brain cells, hepatocytes or testis cells. The method according to any one of claims 1 to 4, wherein the anti-invasive action is a raffle film formation inhibitory action, a cell migration inhibitory action and / or a cell infiltration inhibitory action. An anti-invasive agent comprising N '-(4- (dipropylamino) benzylidene) -2-hydroxybenzohydrazide. The anti-invasive agent according to claim 6, which is an antitumor agent, a cancer cell migration inhibitor, a cancer cell invasion inhibitor, an anti-metastatic agent, or an immunosuppressant.