JP2017075113A - Anticancer agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anticancer agent causing no DNA cleavage.SOLUTION: An anticancer agent comprises a compound represented by the following formula (1) as an active ingredient (in the formula (1), R-Rindependently represent a hydrogen atom, a C1-C30 aliphatic group or a group represented by a formula RCO- (where, R is a C1-C30 aliphatic group or a C1-C10 aromatic group or a hetero aromatic group)).SELECTED DRAWING: None

Description

  The present invention relates to an anticancer agent.

  Topoisomerase is required for DNA synthesis and repair. Topoisomerase is an enzyme that eliminates kinking of DNA. It cleaves a single strand or double strand of DNA and eliminates kinking caused by DNA replication.

  Topoisomerases are classified into topoisomerase I in which DNA cleavage is single-stranded and topoisomerase II in which DNA cleavage is double-stranded.

  A topoisomerase inhibitor is a compound that inhibits the activity of topoisomerase. Topoisomerase inhibitors are classified into DNA-cleaved topoisomerase inhibitors that inhibit topoisomerase activity at the DNA cleavage stage and enzyme-inhibited topoisomerase inhibitors that inhibit topoisomerase activity at stages other than the DNA cleavage stage.

  Since cancer cells actively synthesize DNA, topoisomerase inhibitors are used as anticancer agents. A DNA-cleaving topoisomerase inhibitor induces apoptosis of cancer cells through a DNA damage checkpoint. On the other hand, an enzyme-inhibited topoisomerase inhibitor induces cell division inhibition by a DNA kinking elimination monitoring mechanism. Currently, topoisomerase inhibitors used in anticancer agents are DNA-cleavable.

  By the way, 3EZ, 20Ac-ingenol is a topoisomerase inhibitor, and it has been reported that DNA cleavage is not caused and cell growth of chicken B cell-derived DT40 cells is stopped in the G2 / M phase (for example, non-patented). See reference 1.)

Yasuaki Fukuda, et al., 3EZ, 20Ac-ingenol, a catalytic inhibitor of topoisomerases, downregulates p-Akt and induces DSBs and apoptosis of DT40 cells, Arch. Pharm. Res. (2013) 36: 1029-1038

  The mechanism of action of a DNA-cleaving topoisomerase inhibitor as an anticancer agent utilizes a monitoring mechanism induced by DNA damage, and causes many side effects.

  For example, DNA cleavage that occurs due to treatment with a DNA-cleaving topoisomerase inhibitor may cause secondary cancer after 2 to 3 years.

  In addition, topoisomerase inhibitors tend to induce apoptosis in cells with fast DNA synthesis. For this reason, normal bone marrow hematopoietic stem cells and undifferentiated cells that are rapidly proliferating may decrease, and myelosuppression, leukopenia, and the like may occur.

  It is also known that the heart has high topoisomerase II activity. For this reason, when a topoisomerase inhibitor that inhibits topoisomerase II is administered, many cells undergo apoptosis in the heart, which may cause heart damage.

On the other hand, conventional enzyme-inhibited topoisomerase inhibitors have low cell growth inhibitory activity (IC ₅₀ = 5 to ₅₀ μM), and multinucleated cells may be generated after cell cycle arrest, and these multinucleated cells are secondary cancers. May occur.

  An object of the present invention is to provide an anticancer agent that does not cause DNA cleavage.

The present invention is as follows.
[1] An anticancer agent containing a compound represented by the following formula (1) as an active ingredient.
Wherein ^{(1), R} 1 ~R ¹¹ independently represents a hydrogen atom, RCO- (where the aliphatic group or the formula C1-30, R represents an aliphatic group having 1 to 30 carbon atoms Or represents an aromatic group or heteroaromatic group having 1 to 10 carbon atoms). ]
[2] The anticancer agent according to [1], which is an activator of ATM (Ataxia-telangiectasia mutated) / ATR (Rad3-related).
[3] The anticancer agent according to [1] or [2], which is an Akt phosphorylation inhibitor.
[4] The anticancer agent according to any one of [1] to [3], which is a p53 stabilizer.
[5] The anticancer agent according to any one of [1] to [4], which is an apoptosis inducer.

  According to the present invention, an anticancer agent that does not cause DNA cleavage can be provided.

6 is a graph showing the results of Experimental Example 1. (A)-(e) is a graph which shows the result of the flow cytometry of Experimental example 2. FIG. 6 is a photograph showing the results of Western blotting in Experimental Example 2. 6 is a photograph showing the results of Western blotting in Experimental Example 3. 6 is a photograph showing the results of Western blotting in Experimental Example 4. 10 is a photograph showing the results of Western blotting in Experimental Example 5. 10 is a photograph showing the results of Western blotting in Experimental Example 6. 10 is a photograph showing the results of Western blotting in Experimental Example 7. (A)-(e) is a graph which shows the result of the flow cytometry of Experimental example 7. FIG. 10 is a graph showing the results of Experimental Example 8. 10 is a photograph showing the results of Western blotting in Experimental Example 9. 10 is a photograph showing the results of Western blotting in Experimental Example 9. 10 is a graph showing the results of MTT assay in Experimental Example 9. 6 is a graph showing the results of Experimental Example 1.

[Anticancer agent]
In one embodiment, the present invention provides an anticancer agent containing a compound represented by the following formula (1) as an active ingredient.
Wherein ^{(1), R} 1 ~R ¹¹ independently represents a hydrogen atom, RCO- (where the aliphatic group or the formula C1-30, R represents an aliphatic group having 1 to 30 carbon atoms Or represents an aromatic group or heteroaromatic group having 1 to 10 carbon atoms). ]

  In formula (1), the aliphatic group may be linear, branched, saturated, unsaturated, or substituted. May be unsubstituted. Moreover, 1-20 may be sufficient as carbon number of an aliphatic group, for example, 1-16 may be sufficient as it. Examples of the substituent of the aliphatic group include a halogen atom, a hydroxyl group, an ether group, a carbonyl group, a carboxyl group, an amino group, and an amide group.

  Of the groups represented by the formula RCO- in the formula (1), those in which R is an aliphatic group include acetic acid, propionic acid, butyric acid, 2,3-dimethylbutanoic acid, caprylic acid, capric acid, laurin A group obtained by removing a hydroxyl group from a carboxyl group of a saturated fatty acid having 1 to 16 carbon atoms such as acid, myristic acid and palmitic acid; from a carboxyl group of an unsaturated fatty acid having 1 to 16 carbon atoms such as 2,4-decadienoic acid Examples include a group excluding a hydroxyl group.

  In the formula (1), the aromatic group and the heteroaromatic group may be substituted or unsubstituted. Moreover, 1-8 may be sufficient as carbon number of an aromatic group and heteroaromatic group, for example, 1-6 may be sufficient as it. Examples of the substituent for the aromatic group and heteroaromatic group include a halogen atom, a hydroxyl group, an ether group, a carbonyl group, a carboxyl group, an amino group, an amide group, and the like.

  In the formula (1), among the groups represented by the formula RCO-, those in which R is an aromatic group include a hydroxyl group from a carboxyl group of an aromatic carboxylic acid such as benzoic acid, phthalic acid, salicylic acid, and anthranilic acid. Excluded groups and the like.

  In the formula (1), among the groups represented by the formula RCO-, those in which R is a heteroaromatic group include furan carboxylic acid, thiophene carboxylic acid, pyridine carboxylic acid, nicotinic acid, isonicotinic acid and the like And a group obtained by removing the hydroxyl group from the carboxyl group of the heteroaromatic carboxylic acid.

In the compound represented by the above formula (1), R ² , R ³ , R ⁵ , R ⁶ and R ⁹ are hydrogen atoms, R ⁷ , R ⁸ , R ¹⁰ and R ¹¹ are methyl groups, ¹ may be a group represented by the following formula (2) or (3) or a hydrogen atom, and R ⁴ may be an acetyl group. In the following formulas (2) and (3), R ¹² represents an alkyl group having 1 to 10 carbon atoms.

  The compound represented by the above formula (1) may be 3EZ, 20Ac-ingenol represented by the following formula (4), or may be 3EE, 20Ac-ingenol represented by the following formula (5). Alternatively, 20Ac-ingenol represented by the following formula (6) may be used.

  As will be described later in Examples, the compound represented by the above formula (1) induces apoptosis in cancer cells. Further, even at a low concentration of 0.01 μM, the proliferation of BALL-1 cells, which is an acute leukemia cell line, is significantly suppressed.

  As described in Non-Patent Document 1, the compound represented by the above formula (1) is an enzyme-inhibited topoisomerase inhibitor that does not cause DNA cleavage. Therefore, even when used for a long time, it is difficult for secondary cancer to occur. To date, no enzyme-inhibited topoisomerase inhibitor effective as an anticancer agent has been known.

  In addition, conventional topoisomerase inhibitors tend to induce apoptosis in cells with fast DNA synthesis, and when administered to humans or non-human animals, normal bone marrow hematopoietic stem cells and undifferentiated cells that rapidly proliferate are reduced, and myelosuppression is achieved. In some cases, leukopenia may occur.

  On the other hand, the compound represented by the above formula (1) is considered to induce DNA kinking elimination monitoring mechanism without causing DNA cleavage. And it is known that the DNA kinking elimination monitoring mechanism is deficient in stem cells, undifferentiated cells and the like. Therefore, it can be expected that the compound represented by the above formula (1) does not induce apoptosis of stem cells, undifferentiated cells and the like, and does not cause myelosuppression and leukopenia even when administered to humans or non-human animals. .

  In addition, since the compound represented by the above formula (1) mainly targets topoisomerase I, not topoisomerase II having high activity in the heart, even when administered to humans or non-human animals, Less likely to have side effects.

  From the above, the anticancer agent of this embodiment is useful as an anticancer agent with few side effects. Cancers targeted by the anticancer agent of the present embodiment include PI3 (Phosphoinoside 3-kinase) kinase / Akt pathway activated and phosphorylation of Akt protein increased (intracellular concentration of phosphorylated Akt protein increased) More specifically, leukemia, lymphoma, pancreatic cancer, brain tumor and the like can be mentioned. As will be described later, for example, it is known that PI3 kinase / Akt pathway is activated and phosphorylation of Akt protein is increased in leukemia, lymphoma, pancreatic cancer, brain tumor and the like.

  The anticancer agent of this embodiment may be a pharmaceutical composition containing the compound represented by the above formula (1) and a pharmaceutically acceptable carrier.

  The pharmaceutical composition may be formulated into a dosage form used orally or a dosage form used parenterally. Examples of the dosage form used orally include tablets, capsules, elixirs, microcapsules and the like. Examples of the dosage form used parenterally include injections, suppositories, patches and the like.

  Examples of the pharmaceutically acceptable carrier include solvents such as sterilized water and physiological saline; binders such as gelatin, corn starch, tragacanth gum and gum arabic, excipients such as crystalline cellulose; corn starch, gelatin, alginic acid and the like A swelling agent etc. are mentioned.

  The pharmaceutical composition may contain an additive. Additives include lubricants such as magnesium stearate; sweeteners such as sucrose, lactose and saccharin; flavoring agents such as peppermint and red oil; stabilizers for benzyl alcohol and phenol; buffers such as phosphate and sodium acetate Agents; solubilizing agents such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives; surfactants;

  The pharmaceutical composition can be formulated by combining the above carriers and additives as appropriate and admixing them in a unit dosage form generally required for pharmaceutical practice.

  Examples of the solvent for injection include isotonic solutions containing auxiliary agents such as physiological saline, glucose, D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Solvents for injections may contain alcohols such as ethanol; polyalcohols such as propylene glycol and polyethylene glycol; nonionic surfactants such as polysorbate 80 (trademark) and HCO-50.

  For example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, transdermal, or oral administration to a patient are performed by methods known to those skilled in the art. sell. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose.

  The dose of the compound varies depending on the symptom, but in the case of oral administration, it is generally about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day for an adult (with a body weight of 60 kg). Preferably it is considered to be about 1.0 to 20 mg.

  When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, and administration method. For example, in the form of an injection, it is usually 1 for an adult (with a body weight of 60 kg). It may be convenient to administer about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection.

[ATM / ATR activator]
In one embodiment, the present invention provides an ATM / ATR activator (ATM activator or ATR activator) containing the compound represented by the formula (1) as an active ingredient.

  ATM and ATR are sensors that monitor DNA damage and are factors that work upstream of the DNA damage response pathway.

  As will be described later in Examples, the inventors have revealed that the compound represented by the above formula (1) up-regulates ATM and ATR expression in cells at a low concentration of about 0.5 μM. .

  Therefore, it can be said that the compound represented by the above formula (1) is an activator of ATM or ATR. Here, activation means increasing the function of ATM or ATR, may be increasing the phosphorylation level of ATM or ATR, and increasing the expression of mRNA or protein of ATM or ATR. It may be.

  The ATM / ATR activator of this embodiment is useful as a new research reagent for activating ATM or ATR in cells. Moreover, as shown in the Example mentioned later, the ATM / ATR activator of this embodiment is useful also as a new research reagent which activates ATM or ATR and PTEN in a cell simultaneously. In this specification, the research reagent may be excluded from use as a medicine. It can be said that the compound represented by the above formula (1) is an enzyme-inhibited topoisomerase inhibitor that activates ATM / ATR. To date, there are no known enzyme-inhibited topoisomerase inhibitors that upregulate the expression of ATM / ATR.

  Examples of the cells for which the ATM / ATR activator of the present embodiment activates ATM / ATR include human or non-human animal cells. More specifically, examples include cells in which the PI3 kinase / Akt pathway is activated.

  PTEN is an enzyme that catalyzes the dephosphorylation reaction of phosphatidylinositol 3,4,5-triphosphate, which is an inositol phospholipid. In primary leukemia cells, mutations in the PTEN gene are hardly detected, but it is known that the PI3 kinase / Akt pathway is often activated.

  Here, the activation of the PI3 kinase / Akt pathway means that the hydroxyl group at the 3-position of the inositol ring of the inositol phospholipid is phosphorylated by PI3 kinase and the pathway in which Akt is phosphorylated is promoted. When Akt is phosphorylated and activated, signals that activate the survival and proliferation of various cells are transmitted. In addition, there is a case where MDM2 is phosphorylated by Akt activation and transferred from the cytoplasm to the nucleus, and the activity of p53 is regulated in a regulated manner by decreasing the expression of p53 or the stabilizing effect of p53. . In some cases, activation of Akt may phosphorylate Bad to inhibit apoptosis-inducing activity. It is known that the PI3 kinase gene and the Akt gene are mutated in many types of cancer cells including pancreatic cancer, whereby the PI3 kinase / Akt pathway may be activated.

[Akt phosphorylation inhibitor]
In one embodiment, the present invention provides an Akt phosphorylation inhibitor containing a compound represented by the above formula (1) as an active ingredient.

  As will be described later in Examples, the inventors have clarified that the compound represented by the above formula (1) down-regulates the concentration of phosphorylated Akt in cells at a low concentration of about 0.5 μM. .

  Therefore, it can be said that the compound represented by the above formula (1) is an Akt phosphorylation inhibitor. “Akt phosphorylation inhibitor” can be restated as “Akt activation inhibitor”, “Akt inactivator” and the like. Here, the suppression of Akt phosphorylation may be to activate phosphatidylinositol triphosphate (PIP3) phosphatase, or to reduce the activity of PI3 kinase. Then, dephosphorylation of phosphorylated Akt may be performed, or phosphorylation of Akt may be inhibited.

  The Akt phosphorylation inhibitor of this embodiment is useful as a new research reagent for suppressing phosphorylation of Akt in cells. Moreover, as shown in the Example mentioned later, the Akt phosphorylation inhibitor of this embodiment stabilizes p53 by activation of ATM or ATR in a cell. In addition, it is known that phosphorylation of Akt is also suppressed by a feedback loop due to stabilization of p53. Therefore, the Akt phosphorylation inhibitor of this embodiment suppresses the phosphorylation of Akt dependent on the dephosphorylation activity of PTEN by the activation of PTEN, and stabilizes p53 independent of the dephosphorylation activity of PTEN. It is also useful as a new research reagent that simultaneously stabilizes p53 by activation of ATM or ATR and suppresses phosphorylation of Akt by stabilizing p53.

  Examples of the cells targeted by the PTEN activator of the present embodiment for activating PTEN include those similar to the cells targeted by the ATM / ATR activator described above.

[P53 stabilizer]
In one embodiment, the present invention provides a p53 stabilizer containing a compound represented by the above formula (1) as an active ingredient.

  As will be described later in Examples, the inventors show that the compound represented by the above formula (1) stabilizes p53 in cells at a low concentration of about 0.5 μM and increases the intracellular concentration of p53. Was revealed.

  Therefore, it can be said that the compound represented by the above formula (1) is a p53 stabilizer. Here, stabilization means that the intracellular concentration of p53 is increased, and may be to increase the phosphorylation level or acetylation level of p53, and to increase the expression of p53 mRNA or protein. It may be.

  The p53 stabilizer of this embodiment is useful as a new research reagent for stabilizing p53 in cells.

  Examples of the cells targeted by the p53 stabilizer of this embodiment for stabilizing p53 include the same cells as the cells targeted by the ATM / ATR activator described above.

[Apoptosis inducer]
In one embodiment, the present invention provides an apoptosis-inducing agent (apoptosis-inducing agent) containing the compound represented by the above formula (1) as an active ingredient.

  As will be described later in Examples, the inventors have clarified that the compound represented by the above formula (1) induces apoptosis.

  Therefore, it can be said that the compound represented by the above formula (1) is an apoptosis inducer.

  Examples of the cells targeted by the apoptosis inducer of the present embodiment that induce apoptosis include the same cells as those targeted by the ATM / ATR activator described above.

(Other embodiments)
In one embodiment, the present invention provides a method for treating cancer, comprising the step of administering to a subject a compound represented by the above formula (1).

  In one embodiment, the present invention provides a method for activating ATM / ATR in a cell of a subject, comprising the step of administering a compound represented by the above formula (1) to the subject.

  In one embodiment, the present invention provides a method for suppressing phosphorylation of Akt in a cell of a subject, comprising the step of administering a compound represented by the above formula (1) to the subject.

  In one embodiment, the present invention provides a method for stabilizing p53 in a cell of a subject, comprising the step of administering to the subject a compound represented by the above formula (1).

  In one embodiment, the present invention provides a method for inducing apoptosis in a cell of a subject, comprising administering to the subject a compound represented by the above formula (1).

  In one embodiment, the present invention provides a compound represented by formula (1) above for the treatment of cancer.

  In one embodiment, the present invention provides a compound represented by the above formula (1) for ATM / ATR activation, inhibition of Akt phosphorylation, p53 stabilization or induction of apoptosis.

  In one embodiment, the present invention provides a compound represented by the above formula (1) for the production of an anticancer agent.

  In one embodiment, the present invention provides a compound represented by the above formula (1) for the production of an ATM / ATR activator, an Akt phosphorylation inhibitor, a p53 stabilizer or an apoptosis inducer.

  Next, although an experiment example is shown and this invention is demonstrated in detail, this invention is not limited to the following experiment examples.

[Experiment 1]
(3EZ, 20Ac-ingenol treatment suppressed the growth of BALL-1 cells)
To human breast cancer cell line MCF-7 cell, immortalized human B cell line HEV0011 cell and acute leukemia cell line BALL-1 cell, 3EZ, 20Ac-ingenol was added to 0.01, 0.1,. 5. After 48 hours exposure at a concentration of 1 μM, cell proliferation was examined by MTT assay. As a control, each cell not exposed to the drug was used. FIG. 1 is a graph showing the results.

  As a result, significant growth suppression of BALL-1 cells was observed as compared with control cells. Specifically, in the presence of 0.01 μM 3EZ, 20Ac-Ingenol, the growth of BALL-1 cells was suppressed to about 30% of the control. On the other hand, the growth of MCF-7 cells and HEV0011 cells was not significantly affected by 3EZ, 20Ac-ingenol in the concentration range of 0.01 to 1 μM.

  As shown in the experimental examples described later, phosphorylation of Akt protein is increased in BALL-1 cells, and the intracellular concentration of phosphorylated Akt protein is high, so that 3EZ, 20Ac-ingenol is effectively acted on. Conceivable. In contrast, it has been reported that the presence of phosphorylated Akt protein is not detected in MCF-7 cells. HEV0011 cells are normal immortalized cells.

  Subsequently, the growth inhibitory effect of 3EZ, 20Ac-ingenol was compared among the above-mentioned BALL-1 cells, other leukemia cells, the T cell acute leukemia cell line TKG0377 cell, and the myeloid leukemia cell line TKG0210 cell. After 3EZ, 20Ac-ingenol was exposed to these cells at concentrations of 0.5, 1, 5, 10, 20 μM for 48 hours, cell proliferation was examined by MTT assay. As a control, each cell not exposed to the drug was used. FIG. 14 is a graph showing the results.

  As a result, in the presence of 0.5 μM 3EZ, 20Ac-ingenol, the growth of BALL-1 cells was suppressed to about 40% of the control. On the other hand, the growth of TKG0377 and TKG0210 cells was 80-90% of the control in the concentration range of 0.5-20 μM. That is, these cells were less affected by 3EZ, 20Ac-Ingenol compared to the marked growth inhibition of BALL-1 cells.

[Experiment 2]
(3EZ, 20Ac-ingenol treatment stopped cell growth in S phase)
BALL-1 cells were treated with 0.5 μM 3EZ, 20Ac-Ingenol and the cell cycle was analyzed after 6, 12, 24 and 48 hours using flow cytometry. 2A to 2E are graphs showing the results of flow cytometry analysis. In this analysis, the live cells were gated. As a result, it was clarified that 3EZ, 20Ac-ingenol treatment increased the proportion of S-phase cells to 68% after 6 hours, and to 75% after 12 hours and 24 hours. On the other hand, in the control cells, the proportion of S phase cells remained at 53%. This result shows that the cell growth of BALL-1 cells was stopped in the S phase by treatment with 3EZ, 20Ac-Ingenol.

  Subsequently, BALL-1 cells were treated with 3EZ, 20Ac-ingenol, and changes in the abundance of cyclin A protein known to appear in S phase were examined by Western blotting.

  Specifically, BALL-1 cells were treated with 0.5 μM 3EZ, 20Ac-Ingenol, and Western blotting using an anti-cyclin A antibody (Santa Cruz) was performed after 3, 6, 12, 24 and 48 hours. It was. In addition, an actin protein was detected using an anti-actin antibody (Sigma) as a control. FIG. 3 is a photograph showing the results of Western blotting.

  As a result, it was revealed that the abundance of cyclin A protein was increased by treatment with 0.5 μM of 3EZ, 20Ac-ingenol. This result further reinforces that the cell growth of BALL-1 cells was stopped in the S phase by 3EZ, 20Ac-ingenol treatment.

  As described above, Non-Patent Document 1 reports that 3EZ, 20Ac-ingenol stopped cell growth of DT40 cells in the G2 / M phase. In contrast, S-phase arrest was observed in BALL-1 cells, which revealed that cell growth inhibition completely different from that of DT40 cells occurred.

[Experiment 3]
(3EZ, 20Ac-ingenol treatment induced the expression of ATR and ATM and increased the intracellular concentration of p53 protein)
BALL-1 cells were administered with 3EZ, 20Ac-ingenol to examine whether or not a kinase that monitors DNA damage is activated. Specifically, ATR, ATM, and p53 protein were detected by Western blotting using an anti-ATR antibody (Santa Cruz), an anti-ATM antibody (Millipore), and an anti-p53 antibody (Santa Cruz). In addition, an actin protein was detected using an anti-actin antibody (Sigma) as a control. FIG. 4 is a photograph showing the results of Western blotting.

  As a result, the expression of ATR protein was slightly increased by 3EZ, 20Ac-ingenol treatment for 3 hours, and gradually increased by 3EZ, 20Ac-ingenol treatment for 6 to 12 hours.

  The expression level of ATM protein was also increased by 6 hours of 3EZ, 20Ac-ingenol treatment. Moreover, the intracellular concentration of p53 protein also increased by 3EZ, 20Ac-ingenol treatment for 3 hours and 6 hours, and further increased until 24 hours later. On the other hand, no change was observed in the expression level of the control actin. The increase in intracellular concentration of p53 protein was thought to be due to the stabilization of p53 by phosphorylation by ATM / ATR.

[Experimental Example 4]
(3EZ, 20Ac-Ingenol treatment up-regulated PTEN protein expression and down-regulated Akt protein phosphorylation)
PTEN, a tumor suppressor, is known to negatively regulate PI3K / Akt-dependent pathways. Therefore, 3EZ, 20Ac-ingenol was administered to BALL-1 cells, and changes in the expression level of PTEN protein and changes in the amount of phosphorylated Akt (Ser473) were examined by Western blotting. As antibodies, anti-PTEN antibody (Santa Cruz) and anti-phosphorylated Akt antibody (Ser473, Cell Signaling Technology) were used. In addition, the same study was performed on a sample obtained by administering 10-hydroxycamptothecin, which is a DNA-cleaving topoisomerase inhibitor, to BALL-1 cells. In addition, an actin protein was detected using an anti-actin antibody (Sigma) as a control. FIG. 5 is a photograph showing the results of Western blotting.

  As a result, when treated with 0.5 μM 3EZ, 20Ac-Ingenol for 3 hours, the expression level of PTEN protein in BALL-1 cells was clearly increased compared to untreated control cells. This rise continued until 48 hours later. Furthermore, the time-dependent increase in the expression of PTEN protein inhibited phosphorylated Akt (Ser473) levels over 3 to 48 hours after 3EZ, 20Ac-ingenol treatment.

  PTEN protein was also detected in BALL-1 cells 24 hours after treatment with 0.2 μM 10-hydroxycamptothecin.

  Therefore, it can be said that 3EZ, 20Ac-ingenol is an enzyme-inhibited topoisomerase inhibitor that activates PTEN and down-regulates phosphorylation of Akt protein. To date, there are no known enzyme-inhibited topoisomerase inhibitors that activate PTEN and down-regulate phosphorylation of Akt protein.

[Experimental Example 5]
(3EZ, 20Ac-ingenol treatment induced p21 protein expression)
The p21 protein is one of the targets of the p53 protein that is a transcription factor. Therefore, 3EZ, 20Ac-ingenol was administered to BALL-1 cells, and changes in the expression level of p21 protein were examined by Western blotting. An anti-p21 antibody (Santa Cruz) was used as the antibody. In addition, an actin protein was detected using an anti-actin antibody (Sigma) as a control. FIG. 6 is a photograph showing the results of Western blotting.

  As a result, it became clear that treatment of BALL-1 cells with 0.5 μM of 3EZ, 20Ac-Ingenol for 12 hours induced the expression of p21 protein.

  Induced expression of p21 protein strongly supports the activation of p53 protein. In addition, since p21 protein is also an inhibitor of cyclin A-CDK that promotes DNA synthesis, the induction of p21 protein expression supports S phase arrest.

[Experimental Example 6]
(3EZ, 20Ac-ingenol treatment induced phosphorylation of H2AX protein, increased expression of Bax protein, and decreased expression of Bcl-2 protein)
It has been shown that 3EZ, 20Ac-ingenol treatment does not cause DNA damage. Therefore, whether or not 3EZ, 20Ac-ingenol treatment shows a DNA damage response was examined.

  3EZ, 20Ac-ingenol was administered to BALL-1 cells, and phosphorylation of H2AX protein and changes in expression levels of Bax protein and Bcl-2 protein were examined by Western blotting. As the antibodies, anti-phosphorylated H2AX antibody (γH2AX antibody, Ser139, Millipore), anti-Bax antibody (Cell Signaling Technology), and anti-Bcl-2 antibody (Santa Cruz) were used. In addition, an actin protein was detected using an anti-actin antibody (Sigma) as a control. FIG. 7 is a photograph showing the results of Western blotting.

  As a result, it was revealed that phosphorylation of H2AX protein was induced when BALL-1 cells were treated with 0.5 μM 3EZ, 20Ac-ingenol for 12 hours. This result indicates that 3EZ, 20Ac-ingenol treatment induces a response similar to the DNA damage response.

  By the way, it is known that topoisomerase 1-DNA complex is formed by reactive oxygen species (ROS), and secondary DNA damage is caused by caspase.

  The inventors have also found that cell growth inhibition by 3EZ, 20Ac ingenol is suppressed by caspase inhibition.

  From the above, the response similar to the above-mentioned DNA damage response is not only a direct topoisomerase damage effect by 3EZ, 20Ac-ingenol, but also a secondary damage similar to the DNA damage response induced by the generation of ROS. It may be derived from DNA damage.

  The Bcl-2 / Bax ratio is also known to determine the balance between apoptosis induction and apoptosis inhibition. As shown in FIG. 7, it was revealed that treatment with 0.5 μM 3EZ, 20Ac-ingenol for 24 hours induced an increase in expression of Bax protein. It was also revealed that treatment with 0.5 μM 3EZ, 20Ac-Ingenol for 12 hours induced a decrease in the expression of Bcl-2 protein. From the above results, it was clarified that the induction of apoptosis in BALL-1 cells by 3EZ, 20Ac-ingenol treatment was associated with a marked decrease in the Bcl-2 / Bax ratio.

[Experimental Example 7]
(3EZ, 20Ac-Ingenol treatment induced caspase 3 activation and increased sub-G1 cell population)
In order to examine whether caspase 3 is related to apoptosis induced by 3EZ, 20Ac-ingenol treatment of BALL-1 cells, an antibody specific for cleaved caspase 3 (R & D Systems) Western blotting using was performed. In addition, an actin protein was detected using an anti-actin antibody (Sigma) as a control. FIG. 8 is a photograph showing the results of Western blotting.

  As a result, it was clarified that caspase 3 was activated when treated with 0.5 μM 3EZ, 20Ac-ingenol for 12 hours, and further activated after 24 hours.

  Combined with the reduction in the Bcl-2 / Bax ratio described above, this result shows that caspase-mediated apoptosis occurs in 3EZ, 20Ac-ingenol treated BALL-1 cells.

  Subsequently, apoptosis of BALL-1 cells treated with 3EZ, 20Ac-ingenol was examined by flow cytometry analysis. FIGS. 9A to 9E are graphs showing the results of flow cytometry analysis.

  As a result, apoptosis was observed in about 16% of cells after 24 hours of 3EZ, 20Ac-ingenol treatment, and the ratio increased remarkably after 48 hours, and about 33% of the cells were in the sub-G1 phase where apoptosis occurred. It became clear. On the other hand, in the untreated BALL-1 cells as a control, the proportion of cells in the sub-G1 phase was only about 4.8%.

  From the above results, it was revealed that 3EZ, 20Ac-ingenol induces apoptosis by signal transduction from inhibition of kinking of DNA. Such compounds are not known to date. Therefore, 3EZ, 20Ac-ingenol is considered to be very useful as a research reagent for analyzing a signal transduction pathway that induces apoptosis from inhibition of kinking of DNA.

[Experimental Example 8]
(Examination of compounds similar to 3EZ, 20Ac-ingenol)
BALL-1 cells were exposed to 3EZ, 20Ac-Ingenol, 3EE, 20Ac-Ingenol, 20Ac-Ingenol and Ingenol at concentrations of 0.01, 0.05, 0.1, 0.5 and 1 μM for 48 hours, Cell proliferation was examined by MTT assay. Cells that were not exposed to the drug were used as controls. The chemical formulas of 3EZ, 20Ac-Ingenol, 3EE, 20Ac-Ingenol, and 20Ac-Ingenol are as described above. The chemical formula of ingenol is shown in the following formula (7).

  FIG. 10 is a graph showing the results. As a result, remarkable growth inhibition of BALL-1 cells was observed in the presence of 3EZ, 20Ac-ingenol and 3EE, 20Ac-ingenol. In addition, inhibition of the growth of BALL-1 cells was also observed in the presence of 20Ac-ingenol. On the other hand, ingenol did not significantly affect the growth of BALL-1 cells.

[Experimental Example 9]
(3EZ, 20Ac-ingenol treatment suppressed cell growth by highly expressing ATR protein)
BALL-1 cells were treated with 3EZ, 20Ac-ingenol, and changes in the expression level of ATR were examined. Specifically, 0.5 μM 3EZ, 20Ac-Ingenol was added to the BALL-1 cell medium, and the ATR protein after 12, 24 and 48 hours was subjected to Western blotting using an anti-ATR antibody (Santa Cruz). Detected. As a control, BALL-1 cells not treated with 3EZ, 20Ac-ingenol were used.

  FIG. 11 is a photograph showing the results of Western blotting. As a result, it was revealed that the expression level of ATR protein is increased by the treatment with 3EZ, 20Ac-ingenol.

  Subsequently, ATR expression was knocked down using siRNA against ATR, and the same experiment was performed. Specifically, first, siRNA against ATR (trade name “ON-TARGET plus Human ATR siRNA-SMART pool”, Dharmacon) was added to the culture medium of BALL-1 cells to a final concentration of 50 nM and cultured for 48 hours. Thereafter, 0.5 μM 3EZ, 20Ac-Ingenol was added to the medium, and ATR protein in the cells after culturing for 12, 24 and 48 hours was detected by Western blotting using an anti-ATR antibody (Santa Cruz). As a control, non-targeting siRNA (trade name “ON-TARGET plus Non-targeting pool”, Dharmacon) was added to the medium to a final concentration of 50 nM, and BALL-1 cells cultured for 48 hours were used.

  FIG. 12 is a photograph showing the results of Western blotting. As a result, it was shown that when ATR was knocked down, the expression level of ATR protein decreased with time even when 3EZ, 20Ac-ingenol treatment was performed.

  Subsequently, the effect of ATR knockdown and 3EZ, 20Ac-ingenol treatment on the proliferation of BALL-1 cells was examined. Specifically, BALL-1 cells were cultured for 48 hours in the presence or absence of siRNA against ATR, 0.5 μM 3EZ, 20Ac-ingenol was added to the medium, and the cells were further cultured for 48 hours. Subsequently, cell proliferation was measured by MTT assay. As a control, non-targeting siRNA (trade name “ON-TARGET plus Non-targeting pool”, Dharmacon) was added to the medium to a final concentration of 50 nM, cultured for 48 hours, and further cultured for 48 hours. One cell was used.

  FIG. 13 is a graph showing the results. As a result, the proliferation rate of BALL-1 cells treated with 3EZ, 20Ac-ingenol was reduced to 56% of the control. On the other hand, the growth rate of BALL-1 cells treated with 3EZ, 20Ac-ingenol after knocking down ATR was 77% of the control. This result shows that the growth inhibitory effect by 3EZ, 20Ac-ingenol treatment was partially inhibited by ATR knockdown.

  From the above results, it was revealed that 3EZ, 20Ac-ingenol treatment suppresses cell proliferation by highly expressing ATR protein. This result shows that 3EZ, 20Ac-ingenol treatment also has an anticancer effect independent of PTEN and phosphorylated Akt.

  According to the present invention, an anticancer agent that does not cause DNA cleavage can be provided. In addition, an ATM / ATR activator, an Akt phosphorylation inhibitor, a p53 stabilizer, and an apoptosis inducer that do not cause DNA cleavage can be provided.

Claims (5)

The anticancer agent which contains the compound represented by following formula (1) as an active ingredient.
Wherein ^{(1), R} 1 ~R ¹¹ independently represents a hydrogen atom, RCO- (where the aliphatic group or the formula C1-30, R represents an aliphatic group having 1 to 30 carbon atoms Or represents an aromatic group or heteroaromatic group having 1 to 10 carbon atoms). ]   The anticancer agent according to claim 1, which is an ATM / ATR activator.   The anticancer agent according to claim 1 or 2, which is an Akt phosphorylation inhibitor.   The anticancer agent according to any one of claims 1 to 3, which is a p53 stabilizer.   The anticancer agent according to any one of claims 1 to 4, which is an apoptosis inducer.