JPH11269065A - Controlling agent for apoptosis resistance of cancer cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain medicines for antitumor agent capable alternating human solid carcinoma cells resistant to apoptosis into apoptosis sensitive to an antitumor agent such as adriamycin having action of inducing apoptosis to a certain kind of cancer cells by including 2,5-dihyroxycinnamic acid ethyl ester as an active component. SOLUTION: The medicines include 2,5-dihyroxycinnamic acid ethyl ester of the formula as an active component. The medicines can be prepared by blending the compound of the formula which is the active component, with conventional solids or liquid carriers or a composition form. The medicines can be prepared into a formulation suitable for oral administration or intravenous administration. The compounds of the formula are obtained by reacting ethyl bromoacetate with triphenylphosphine obtaining (carboethoxy methyl) triphenylphosphonium.bromide, subsequently reacting 1N-NaOH aqueous solution, reacting the reaction product with 2,5-dihydroxy-benzaldehyde.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an agent for overcoming cancer cell apoptosis resistance, which has the effect of modifying a cancer cell of the type having apoptosis resistance to an anticancer agent having the effect of inducing apoptosis in cancer cells to apoptosis sensitivity. That is, the present invention relates to an agent for overcoming or reducing cancer cell apoptosis resistance to an anticancer agent.

[0002]

2. Description of the Related Art In Japan, the death toll of cancer per year reaches about 200,000, and cancer is the leading cause of death.
Cancer is a disease of cell abnormalities that occurs when normal cells mutate at the gene level and acquire abnormal growth and metastatic potential. Surgery, chemotherapy, immunotherapy and the like are performed for the treatment of cancer, but the effect of the treatment is limited, and particularly in the case of advanced cancer, treatment has hardly been achieved. For example, in chemotherapy, administration of an anticancer agent such as adriamycin may be effective in treating leukemia, but all existing anticancer agents often do not show a significant effect in treating solid cancer such as pancreatic cancer. .

[0003] New chemotherapeutic agents that are effective against such solid tumors have been sought for many years by many research institutions, but have been without satisfactory success. Therefore,
There is a need for new chemotherapy for solid cancer by a new method utilizing the properties of the solid cancer itself, and effective new drugs to be used therefor.

[0004]

On the other hand, apoptosis was reported as a new concept of cell death in the 1970s [Int.
Rev. Cytol. 68, 251 (1980)]. Compared to the previously known concept of cell death, necrosis, apoptosis takes less time than necrosis after giving a stimulus to cause cell death, and the surrounding tissues around cancer. It does not cause inflammation. Subsequently, it was reported that many anticancer drugs such as adriamycin and cisplatin have an effect of inducing apoptosis in some cultured cells [Cancer Res. 53
Volume 1845 (1993) and Exp.CellRes. 211 Volume 231 (19
94)].

[0005] However, these anticancer drugs such as adriamycin and cisplatin are not effective against leukemia cells or experimental cancer cells artificially produced with a cancer virus or the like.
It has also been found that it induces apoptosis of cells, but does not induce apoptosis in many naturally occurring human solid cancer cells [Clinician, 21, No. 9, pp. 42-45 (199).
Five)〕. Therefore, these conventional anticancer drugs have a property that apoptosis is not induced by these anticancer drugs, that is, a new apoptosis-resistant human solid cancer cell has a property of modifying apoptosis-sensitive human solid cancer cells. If a drug is newly discovered and administered together with an existing anticancer drug to a patient with solid cancer, it is expected that the conventional anticancer drug will be effective for solid cancer cells. .

[0006] It is an object of the present invention to convert a kind of human solid cancer cells having the property of being resistant to apoptosis to an anticancer agent having an effect of inducing apoptosis of certain cancer cells such as adriamycin, It is an object of the present invention to provide a new drug having a pharmacological action that can be modified into a sensitive property.

[0007]

In order to achieve the above object of the present invention, the present inventors have conducted various studies. As a result, in the process of examining the pharmacological action of 2,5-dihydroxycinnamic acid ethyl ester, which is known as a less toxic tyrosine kinase inhibitor, the present inventors have now discovered that it is apoptotic resistant to adriamycin. Human pancreatic cancer cells AsPC-1 cells in vitro
In tests it was found that cell death by apoptosis could be induced when treated with both adriamycin and 2,5-dihydroxycinnamic acid ethyl ester in combination.
In this case, it was also recognized that even if the AsPC-1 cells were treated with 2,5-dihydroxycinnamic acid ethyl ester alone, cell death by apoptosis could not be induced at all.

Accordingly, in the present invention, the following equation An agent for overcoming apoptosis resistance of cancer cells to an anticancer agent, comprising 2,5-dihydroxycinnamic acid ethyl ester represented by the formula:

The agent for overcoming apoptosis resistance of cancer cells according to the present invention comprises a compound of the above formula (A) as an active ingredient, which is commonly used in a solid or liquid carrier such as starch,
It can be formulated into a composition that is admixed with glucose, water, saline, and ethanol. Then, it can be formulated into a known dosage form suitable for oral administration or intravenous administration.

The compound of formula (A), ie, 2,5-dihydroxycinnamic acid ethyl ester, has the effect of modifying cancer cells of the type which are resistant to apoptosis to an anticancer drug such as adriamycin, to properties which are susceptible to apoptosis. As mentioned above, this effect is completely unexpected. This is because this action does not show the corresponding 2,5-dihydroxycinnamic acid butyl ester or hexyl ester.

The agent for overcoming apoptosis resistance of the present invention can also be regarded as an agent for eliminating or reducing the apoptosis resistance of a type of cancer cell having an apoptosis resistance to an anticancer agent. It is a cancer therapeutic agent that can overcome or reduce the apoptosis resistance of cancer cells to the anticancer agent in the treatment of cancer caused by cancer cells that hardly cause apoptosis of cancer cells by administration of the anticancer agent, such as pancreatic cancer. When the agent of the present invention is administered in combination with an anticancer agent, it can overcome or reduce the apoptosis resistance of cancer cells, and thus can exert a therapeutic effect on solid cancer patients.

The compound of the above formula (A) is a compound synthesized as a tyrosine kinase inhibitor [J. Antibiot.
5, p. 280 (1992)), and the EG of human epithelial cancer A431 cells.
F50 tyrosine kinase with an IC ₅₀ concentration of 0.17μ
It has an enzyme inhibitory activity of inhibiting at g / ml (0.81 μM).

[0013] Human pancreatic cancer cells AsPC-1 cells are referred to as "In Vitro.
Vol. 24, p. 1982), which shows extremely strong apoptotic resistance to anti-cancer drugs such as adriamycin, while another human pancreatic cancer cell BxPC
-3 cells (described in Cancer Investigation, 4: 15 (1986)) are susceptible to apoptosis by adriamycin. For example, it has now been found that adriamycin induces apoptosis in BxPC-3 cells at a concentration of 3 μg / ml within 24 hours, but does not induce apoptosis in AsPC-1 cells even at a concentration of 30 μg / ml. Because adriamycin is showing the inhibitory effect of inhibitory effect and cell cycle of cell proliferation IC ₅₀ concentration on the both cells at 0.06-0.07 μg / ml, the resistance of AsPC-1 cells to adriamycin by generally observed drug efflux Apoptosis resistance, not drug resistance. The present inventors have now discovered that treating AsPC-1 cells with a compound of formula (A) together with adriamycin allows apoptosis to be induced by adriamycin.

The method for producing the compound of the formula (A) used as an active ingredient in the overcoming agent of the present invention is described in "J. Antibio
t. ", Vol. 45, p. 280 (1992). Next, the following Reference Example 1 shows a production example of the compound of the formula (A) starting from ethyl bromoacetate.

Reference Example 1 A commercial product (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as ethyl bromoacetate. 4 ml of this ethyl bromoacetate and 9.
After reacting 47 g, 16.94 g of a white powder of (carboethoxymethyl) triphenylphosphonium bromide (yield 10
0%). Next, 16.94 g of this compound was dissolved in 200 ml of water with stirring, a drop of phenolphthalein solution for pH indication was added, and a 1N aqueous solution of NaOH was added until the water in the reaction system turned red. A white precipitate formed as the reaction proceeded. The reaction system was extracted three times with 200 ml of ethyl acetate, the organic layer as the extract was dehydrated with sodium sulfate, filtered through cotton, and the filtrate was concentrated to obtain 11.10 g of a white solid of ethyl triphenylphosphoranylideneacetate ( (Yield 88.44%).

By reacting 0.2301 g of this white solid with 0.0913 g of 2,5-dihydroxybenzaldehyde, ethyl 2,5-dihydroxycinnamic acid ethyl ester of a yellow solid was obtained.
0.0858 g (62.4% yield) was obtained. This product is a yellow solid and has the molecular formula: C ₁₁ H ₁₂ O ₄ , molecular weight 208.21,
The Rf value was 0.48 (developed with chloroform / methanol = 10/1) by silica gel thin layer chromatography.

Next, a test of the modifying effect of the compound of the formula (A) on cancer cells of a type that is resistant to apoptosis to an anticancer drug is shown in the following Test Example 1.

Test Example 1 Human pancreatic cancer cell AsPC was used as a cell line to test the effect of the compound of formula (A) on the effect of overcoming the apoptosis resistance of cancer cells to the anticancer drug adriamycin.
-1 was used. The apoptosis resistance of these cells to adriamycin has now been discovered by the present inventors.

(I) Drug treatment of cancer cells for evaluation of induction of apoptosis Human pancreatic cancer AsPC-1 cells were placed in a culture solution at a rate of 1 × 10 ⁵ cells / ml in a volume of 1 ml, and a cover glass was previously placed on the plate. The plate was sowed on a placed 12-well plastic plate and cultured for one day. After the cancer cells were treated with the drug by adding a solution of the test substance to be tested to the culture solution in the plate, 48 hours later, the culture solution was removed. In the aforementioned drug treatment, 3 μg / ml of adriamycin (adriamycin solution in which adriamycin was once dissolved in DMSO and then diluted with water) alone or as a tyrosine kinase inhibitor was used as the test substance.
using erbstatin alone, or 10 μg / ml of 2,5-dihydroxycinnamic acid ethyl ester, butyl ester or hexyl ester (methanol solution) alone,
Alternatively, drug treatment was carried out using adriamycin in combination with erbstatin or the latter ester.

(Ii) Evaluation of apoptosis 24 hours after the drug treatment, 1 ml of formalin was added to the treated cells, and the cells were immobilized by standing at 4 ° C for 15 minutes. Thereafter buffer ^{PBS - (NaCl 8.0g / l,} KCl 0.2g /
_{l, Na 2 HPO 4 H 2} O 2.31g / l, the cells were washed once with _{KH 2 PO 4 0.2g / l)} , PBS containing Hoechst 33258 dye 10 [mu] g / ml ^-
Was added, and the cells were stained by allowing them to stand in a dark place for 5 minutes. After this dyeing process, PBS ^- with washed once, remove the cover glass, the stained cells topped transferred onto glass slides placed a drop of 50% aqueous glycerol and observed under a fluorescence microscope. At this time, the number of cells in one visual field of the fluorescence microscope was about 100. Among the stained cells, cells that had undergone nuclear condensation or fragmentation were regarded as cells that had been induced to undergo apoptosis.

Adriamycin and compounds of formula (A)
48 hours after drug treatment with both (abbreviation: 2,5-EtC), the percentage value of the total number of stained cells observed under a fluorescence microscope was calculated as apoptosis was calculated under the fluorescence microscope. Also, for the test group in which cells were treated with adriamycin alone, the value of percentage of the total number of cells observed to have undergone apoptosis was calculated. further,
As a comparative drug, a test group in which cells were treated with erbstatin alone or 2,5-dihydroxycinnamic acid ethyl ester (2,5-EtC) alone, and 2,5-dihydroxycinnamic acid butyl ester (abbreviation: 2,5 -BuC) alone or 2,5-dihydroxycinnamic acid hexyl ester (abbreviation:
Similarly, the percentage of the number of cells that had undergone apoptosis was also determined for the test group in which the cells were treated alone with (2,5-HeC).
The value (relative to the total cell number) was calculated. Furthermore, the percentage of the number of apoptotic cells was similarly calculated for a test group in which cells were treated with one of each of these comparative drugs in combination with adriamycin. The same test was performed when BxPC-3 cells were treated with adriamycin alone in place of AsPC-1 cells.

The results obtained are summarized in Table 1 below.

As can be seen from Table 1 above, AsPC-1
For cells, erbstatin, a known tyrosine kinase inhibitor when used in combination with adriamycin, induced some apoptosis, whereas the active ingredient compound 2,5-EtC of the present invention when used in combination with adriamycin was cancerous. cell
Apoptosis was strongly induced by AsPC-1. 2,5
-BuC and 2,5-HeC had no effect. Bx
When PC-3 cells were treated with the test substance alone, the cells peeled off and floated within 48 hours thereafter. Thus, treatment with adriamycin alone for 24 hours induced apoptosis even with adriamycin alone.

(Iii) Suppression of cell growth AsPC-1 cells or BxPC-3 cells cultured in RPMI1640 medium containing 10% FBS are seeded on a 48-well plastic plate at a rate of 3 × 10 ³ cells / well. One day later, the cells were treated with adriamycin. From adriamycin addition
The number of viable cells was counted after incubating the cells for 48 hours. The cell growth rate was calculated on the basis that the number of cells (about 1.3 × 10 ⁴ ) in the control test group to which no drug was added was 100%. With this criterion, cell growth should be 50
The% inhibitory adriamycin concentration (IC ₅₀ value) was determined.

The results are shown in Table 2 below.

As shown in the results of Table 2 above, in AsPC-1 cells, the cell proliferation was inhibited by adriamycin to a similar extent and inhibited as compared with the apoptosis-sensitive human pancreatic cancer cell BxPC-3. You. Apoptosis is easily induced by 3 μg / ml of adriamycin in BxPC-3 cells, but not at all in AsPC-1 cells. But As
2,5-Et in addition to adriamycin in PC-1 cells
Apoptosis began to occur when treated with C.

Therefore, in the case of pancreatic cancer, which is considered to be almost impossible to treat clinically, the use of the present invention can improve the action of anticancer agents such as adriamycin, so that there is a possibility of treating certain types of pancreatic cancer. It is suggested that it was obtained.

Claims (1)

[Claims] 1. The following formula (A) An agent for overcoming apoptosis resistance of cancer cells to an anticancer agent, which comprises 2,5-dihydroxycinnamic acid ethyl ester represented by the formula: