WO2002102814A1

WO2002102814A1 - Apoptosis inducer

Info

Publication number: WO2002102814A1
Application number: PCT/KR2002/001123
Authority: WO
Inventors: Il-Ju Bae; Chang-Hun Rhee; Kang-Moon Seo; In-Cheol Park; Myoung-Jin Park
Original assignee: Individual
Current assignee: Individual
Priority date: 2001-06-16
Filing date: 2002-06-14
Publication date: 2002-12-27
Anticipated expiration: 2003-12-16
Also published as: KR20020095835A

Abstract

The present invention relates to a novel apoptosis-inducer. The present invention, more specifically, relates to an apoptosis-inducer consisting of As4O6 and pharmaceutical compositions thereof. The apoptosis-inducer according to the present invention induces cell death in solid tumors and diverse blood tumors comprising leukemia, and so can be used as a treatment for these tumors.

Description

APOPTOSIS INDUCER

BACKGROUND OF THE INVENTION

1. Fields of the Invention The present invention generally relates to new uses and pharmaceutical compositions of solid As₄0₆ as apoptosis inducers and anti-cancer drugs. More specifically, a novel use of solid As₄0₆ known as "Chunjisan" is disclosed which induces active cell death by apoptosis to treat blood cancer such as leukemia.

2. Description of the Prior Art

Anti-cancer drugs have been developed before the study of apoptosis. And it is recently suggested that an anti-cancer drug induces apoptosis of cancer cells.

However, the mechanism of apoptosis by anti-cancer drugs has not been accurately revealed and it is very important to identify the anti-cancer drug induced apoptosis mechanism as well as develop novel anti-cancer drugs because of the conventional anti-cancer drugs resistant cell and toxicity of anti-cancer drugs.

Cell death proceeds by one of two mechanisms: necrosis or apoptosis. Necrosis is a passive cell degradation process by stimuli such as burn, ischemia and poisonous substance and generates inflammation by releasing cytosolic materials caused by swelling and lysis of cell. Unlike necrosis, apoptosis is a active cell suicide mechanism which results in cell shrinkage, nuclear condensation and DNA fragmentation and does not generate inflammatory response in vivo because apoptotic body eliminates these materials by phagocytosis. The apoptosis is essential for development;, differentiation and homeostasis of multicellular organism.

In the past, arsenic compound had been used as anti-cancer drugs in Oriental medicine. In 1970s, it was confirmed that principal component of arsenical compounds is As203. Recently, it is shown that injection of arsenic trioxide with clinically applicable density (0.5-2μmol/L) had an excellent effect in treating acute promyelocytic leukemia resistive to all-trans retinoic acid (Gallagher, R. E. Arsenic: New life for an old potion. N. Engl. J. Med., 339: 1389-1391, 1998, Shen, S. X., Chen, G. Q, Ni, J. PL, Li, X. S., Xiong, S. M., Qui, Q. Y., Zhu, J., Tang, W., Sun, G. L., Hang, K. Q., Chen, Y., Zhou, L., Fang, Z. W, Wang, Y. T., Ma, J., Zhang, P., Zhang, T. D., Chen, S. J., Chen, Z., and Wang, Z. Y. Use of arsenic trioxide As2O3 in the treatment of acute promyelocytic leukemia (APL). II . Clinical efficacy and pharmacokinetics in relapsed patients. Blood, 89: 3354- 3360, 1997. Soignet, S. L., Maslak P., Wang, Z. G., Jhanwar, S., Calleja, E., Dardashti, L. J., Corso, D., Deblasio, A., Gabrilove, J., Scheinberg, D. A., Pandolfi, P. P., and Warrell, R. Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N. Engl. J. Med., 339: 1341-1348, 1998). Although its medicinal effect mechanism has not been accurately identified, it is reported that the effect results from reduced expression of Bcl-2 and apoptosis induction by destruction of PML-RAR α protein (Chen, G. Q., Zhu, J., Shi, X. G., Ni, J. H., Zhong, H. J., Si, G. Y., Jin, X. L., Tang, W., Li, X. S., Xiong, S. M., Shen, Z. X., Sun, G. L., Ma, J., Zhang, T. D., Gazin, C, Naoe, T., Chen, S. J., Wang, Z. Y., and Chen, Z. In vitro studies on cellular and molecular mechanisms of arsenic trioxide As203 in the treatment of acute promyelocytic leukemia: As203 induces NB4 cell apoptosis with down-regulation of Bcl-2 expression and modulation of PML-RAR/PML proteins. Blood, 88: 1052-1061, 1996). The success in the treatment of acute promyelocytic leukemia by arsenic compound is a landmark of a new anti-cancer drug. Now, arsenic compound is tried to apply in other blood cancers and solid cancers.

However, there are some problems in treating leukemia patients with the arsenic compound such as As203. For examples, in case of the long-term treatment resistant cells appear (Gianni, M., Koken, M. H. M., Chelbi-Alix, M. K.,Benoit, G., Lanotte, M., Chen, Z., de The H. combined arsenic and retinoic acid treatment enhances differentiation and apoptosis in arsenic-resistant NB4 cells. Blood, 91 :4300-4310, 1998), and the treatment effect is weak in leukemia patients who have no fusion proteins such as PML-RAR α (Jing, Y., Dai, J., Chalmers- Redman, R. M. E., Tatton, W. G., Waxman, S. Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hyfrogen peroxide-dependent pathway. Blood, 94:2102-2111, 1999, Wang, Z. G., Rivi, R., Delva, L.„ Konig, A, Scheinberg, D. A., Gambacorti-Passerini, C, Gabrilove, J. L., Warrell, R. P. Jr., Pandolfi, P. P. arsenic trioxide and melarsoprof induce programmed cell death in myeloid leukemia cell lines and function in a PML and PML-RAR independent manner. Blood, 92: 1497-1504, 1998).

Tetraarsenic hexaoxide(solid As₄O₆) has been used as basic material to produce other arsenic compounds like As203, for insecticides, decolorizers in glass manufacture or preservatives of leathery. Like arsenic trioxide, solid As₄0₆ has been known as toxic substance to induce cancers, and chemists focus on only its chemical structure study(Becker K. A., Plieth K., and Stranski I. N. The polymorphic modifications of arsenic trioxide. Prog. Inorg. Chem. 4, 1-72 (1962), Pupp C, Lao R. C, Murray J. J., pottie R. F., Equilibrium vapor concentrations of some polycyclic aromatic hydrocarbons, As₄0₆ and Se02 and the collection efficiencies of these air pollutants. Atomspheric Environment Vol, 8, pp 915-925, 1974, Grzechnik A. Compressibility and vibrational modes in solid As₄0₆ Journal of Solid State Chemistry. 144(2) -.416-422, 1999 May).

The present inventor identified anti-cancer effect of solid As₄0₆ separated and purified from natural arsenic tri oxides and filed a patent application in

Korea(Korean Patent Application No. 1998-164860) and obtained a granted patent on use of solid As₄O₆ as anti-cancer drug in Japan(Japanese Patent No. 3007627) in 1999.

Particularly, it has been reported that oral administration of arsenic trioxide (As203) may cause serious gastrointestinal and hepatic side effects (Shen ZX, Chen GQ, Ni JH, Li XS, Xiong SM, Qiu QY, Zhu J, Tang W, Sun GL, Yang KQ, Chen Y, Zhou L, Fang ZW, Wagn YT, Ma J, Zhang P, Zhang TD, Chen SJ, Chen. Z, Wang ZY Use of arsenic trioxide(As2O3) in the treatment of acute promyelocytic leukemia (APL): JJ . Clinical efficacy and pharmacokinetics in relapsed patients. Blood 1997 May l;89(9):3354-60). However, the present invention is disclosed that tetraarsenic hexoxide (As₄O₆) does not cause gastrointestinal and hepatic side effects but minute histologic change in kidneys when about lOOmg/kg of As₄O₆ is oral-administered to about 200g Sprague- Dawley rat. And the present invention is disclosed that in case of oral- administration of lOmg/kg and lmg/kg of As₄O₆, tetraarsenic hexaoxide causes no gastrointestinal and hepatic side effects.

SUMMARY OF THE INVENTION

Accordingly, the present invention has an object to provide an apoptosis inducer consisting of As₄0₆ compound for treating diverse blood cancers including leukemia and solid tumors.

In order to accomplish the above-described object, the present inventor identifies that tetraarsenic hexaoxide As₄0₆ shown in Fig. 1 induces apoptosis in relatively low concentration to U937 leukemia cell line. By DNA fragmentation and DNA staining with FITC-annexin V, the present inventors identify that tetraarsenic hexaoxide As₄0₆ induces release of Cytochrome c from mitochondrial membrane and activates caspase, thereby inducing apoptosis. The apoptosis mechanism is completely inhibited when generation of reactive oxygen, particularly hydrogen peroxide, is suppressed. From 30 minutes after treatment of As₄O₆, the generation of hydrogen peroxide increases. Accordingly, As₄0₆ of the present invention may induce apoptosis by causing generation of reactive oxygen including generation of excessive hydrogen peroxide from cancer cells, thereby treating diverse blood cancers including leukemia. The U937 leukemia cell is a cell line resistant to the conventional As2O3 with effect of treating leukemia as well as a cell line that do not express fusion protein PML-RAR α . As a result, As₄0₆ of the present invention is effective for treatment of leukemia patients as well as PML patients.

A method of manufacturing the As₄0₆ of the present invention known as "Chunjisan" is disclosed in Korean Patent Application No. 1998-16486. In brief, the As₄0₆ of the present invention can be manufactured through multi-step heating natural arsenic new stone and reagent arsenic. Here, a detail method of manufactured As₄0₆ of the present invention will be omitted.

The As₄0₆ of the present invention manufactured by other methods besides the above-described method may be used if it has the structure of Fig. 1. The dose of As₄0₆ of the present invention may be changed depending on kinds of disease, age, gender, health condition of patients when applied to the human body.

The As₄0₆ of the present invention may be singly used or with other anti- cancer drug and complex preparations. The As₄0₆ of the present invention may also be made into various oral or non-oral preparations such as powders, tablets and liquors by mixing various pharmaceutically acceptable additives such as diluent, disintegrator, air freshener and glidant.

The As₄0₆ of the present invention may be applied to diverse apoptosis related disease, such as blood cancers including leukemia and solid tumors. Here, the As₄O₆ of the present invention may be used with other preparations.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a structural formula of apoptosis inducer of the present invention. Figs. 2a and 2b are graphs showing the effects of apoptosis inducer measured by flow cytometry of the present invention.

Fig. 2c is a picture showing DNA fragmentation in relation to the effects of apoptosis inducer of the present invention.

Fig. 3 is a graph showing mitochondria membrane potential depletion in relation to the effects of apoptosis inducer of the present invention.

Fig. 4 is a Western blotting picture showing Cytochrome c release in cytoplasm in relation to the effects of apoptosis inducer of the present invention.

Figs. 5a and 5b are graphs and pictures showing catapase-3 activation measured by flow cytometry in relation to the effects of apoptosis inducer of the present invention. Fig. 5c is a Western blotting analysis picture.

Fig. 6 is a graph showing dependency of catapase-3 activation measured by flow cytometry in relation to the effects of apoptosis inducer of the present invention. Figs. 7a and 7b are graphs showing apoptosis inhibition effects by antioxidant measured by flow cytometry in relation to the effects of apoptosis inducer of the present invention.

Fig. 7c is a graph showing experimental results of identifying hydrogen peroxide generation in relation to the effects of apoptosis inducer of the present invention.

Fig. 7d is a graph showing blocking apoptosis by catalase measured by flow cytometry in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The disclosed apoptosis inducer will be described in more details referring to examples below, when are not intended to be limiting.

Example 1 : Experiment of measuring apoptosis

The experiment was performed for identifying effects inducing apoptosis of As₄0₆ accordmg to the present invention. Reagents and cells used in examples were obtained according to the following methods.

1) Reagent and cell culture

Tetraarsenic hexaoxide (As₄0₆) provided from Chunjisan Co. (Seoul, Korea) was dissolved in NaOH solution (IM), diluted and. then used in the experiment. Z-DEVD-FMK and N-acetyl-N-cysteine (NAC) provided from Calbiochem (SanDiego, CA, USA), catalase from Sigma Co., and caspase-3 and cytochrome c antibody from Pharmingen (San Diego, CA, USA) were used in Western blotting analysis. 3,3 '-dihexyloxacarbocyanine iodide for observing mitochondria membrane potential depletion and 2',7'-dichlorodihydrofluorescein diacetate for observing generation of hydrogen peroxide were provided from Molecular Probes (Eugene, OR, USA).

U937 cell line, myelogenous leukemia cell, acquired from The Korean Cell Line Bank (Cancer Research Center, Seoul National Medical College, Seoul, - Korea), were successively cultured in culture medium of 5% CO2 at 37°C humidified with RPMI-1640 culture (GIBCO) supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY, USA) and penicillin /streptomycin, and then used in the experiment.

In order to identify apoptosis effects of the tetraarsenic hexaoxide (As O₆) according to the present invention, the present inventors add constant concentration of As₄O₆ to U937 cell line and incubate in RPMI-1640 medium supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY, USA) and penicillin/streptomycin at 5% CO2 and 37°C in the examples below. Hereinafter, this process will be referred to "treatment of As₄O₆".

2) Measurement of apoptosis

After 1 X 105 U937 cells were treated with 0, 0.5, 1.0, 2.5, or 5.0μ M of As₄0₆ and were cultured for 18 hours, the cultured U937 were washed with PBS to observe apoptosis and were suspended with binding buffer solution of lOmM HEPES/NaOH(pH 7.4), 140mM NaCl, 2.5mM CaC12. Then, 5μ 1 of annexin V-FITC and propidium iodide PI(PharMingen, SanDiego, CA, USA) were added in U937 cells and were carried out reaction at room temperature for 15 minutes. The result was measured by flow cytometry and shown in graphs of Figs. 2a and 2b. As shown in Fig. 2a, it was shown that induction of apoptosis by As₄0₆ was concentration-dependent at 0.5~5μ M concentration. Additionally, as shown in Fig. 2b, while annexin V-FITC/PI staining was observed in 5% of cells in control group, 53% of cells were stained in lμ M As₄O₆. As a result, it was shown that more cells were died in As₄0₆.

3) Analysis of DNA fragmentation

After 1 X 105 cells were treated with 0, 0.5, 1, 2.5,

M of As₄O₆ and washed with PBS, the cells were treated with lysis buffer solution consisting of lOmM tris, pH 8.0, lmM EDTA, 1% SDS. Cell homogenate was treated with DNase-free proteinase K and the reaction carried out at 55°C for 1 hour. The resulting mixture was treated with phenol/chloroform. DNAs were precipitated with 5M NaCl and isopropyl alcohol, and then dissolved in distilled water. RNAs were removed using DNase-free RNase (10 units). Electrophoresis was performed on the above resultant in 1.8% agarose gel, and stained with ethylbromide (C2H5Br). Then, DNAs were identified in UV.

As a result, as shown in Fig. 2c, DNA fragmentation was observed at lμ M As₄0₆.

Accordingly, from the experimental results of Example 1, it was shown that As₄0₆ induced apoptosis. Example 2: Measurement of mitochondria membrane potential depletion

As known that the process of inducing apoptosis was closely related to mitochondria, in order to identify whether As₄0₆ induces mitochondria membrane potential depletion in apoptosis mechanism, U937 cells were treated with 2.5μ M As₄O₆ and then the degree of mitochondria membrane potential depletion was observed hourly.

After 1 X 105 U937 cells were treated with 2.5μ M As₄0₆ 3,3 '- dihexyloxacarbocyanine iodide, fluorescent material accumulated in mitochondria, was added at 50nM in the cell culture, and then carried out reaction at 37°C for 15 minutes. The cell culture medium was washed with PBS and analyzed using flow cytometry. The results measured hourly were shown in Fig. 3.

Referring to Fig. 3, six hours after 2.5μ M As₄O₆ was added, mitochondria membrane potential depletion was observed.

Example 3 : Measurement of cytochrome c release in cytoplasm

It had been reported that if mitochondria membrane is damaged due to mitochondrial membrane potential depletion, Cytochrome c was released, thus triggering apoptosis signaling. The experiment was performed for identifying that apoptosis induction of As₄0₆ is related to cytochrome c release in cytoplasm. The 1 X 105 U937 cells were treated with 2.5μ M As₄0₆ for constant time and centrifuged at 1200 rpm for 5 minutes to obtain cell pellets. After the cell pellets were dissolved in solution including 220mM mannitol, 70mM sucrose, 50mM Pipes -KOH (pH 7.4), 50mM KC1, 5mM EGTA, 2mM MgC12, lrnM EDTA, lmM dithiothreitol and various protease inhibitors, the dissolved cell pellets were homogenized and centrifuged, supernatant of the solution was obtained to perform a Western blotting analysis.

10-12% SDS-PAGE(sodium dodecyl sulfate-polyacrylamide electrophoresis) was performed on the Western blotting samples in Tris-glycine running buffer, and then another electrophoresis was performed on the samples in 0.45μ M protron nitrocellulose (Schleicher & Schuell, Germany). The electrophoresis membrane was blocked in blocking buffer solution (5% skim milk) for 30 minutes, and a primary antibody of protein for analysis was diluted in blocking buffer solution at a ratio of 1 : 1,000 and carried out reaction for 1 hour. The membrane combined with the primary antibody was three times washed using PBST for 20 minutes. A secondary antibody conjugated with horse-radish peroxidase was diluted in blocking buffer solution at a ratio of 1 : 3,000 and then carried out reaction for 30 minutes. After reaction, the secondary antibody was three times washed using PBST for 20 minutes, and then stained using ECL system (Amershame, Arlington Heights, IL, USA). The result picture was shown in Fig. 4.

As shown in Fig. 4, after treatment of 2.5μ M As₄0₆, Cytochrome c in cytoplasm was observed. From this experimental result, it was shown that As₄O₆ for inducing apoptosis was related to Cytochrome c release caused by mitochondrial membrane potential depletion.

Example 4: Activation of catapase-3 1) Measurement of catapase-3 activation

In order to identify whether activation of catapase-3 is involved in apoptosis induced by As₄0₆, FAM-DEVD-FMK, Z-DEVD-FMK derivative emitting fluorescence by covalent bond with activated catapase-3, was added in cell culture medium treated with As₄0₆ and fluorescence was detected.

CaspaTag™ catapase activation kit (Intergen, Purchase, NY, USA) was used as a reagent for measuring activation of catapase-3. According to protocol of manufacture, 1 X 105 cells were treated with 2.5μ M As₄0₆ for 18 hours, and lOμ 1 FAM-DEVD-FMK was added in culture solution and carried out reaction in culture medium for 1 hour. After the culture medium was washed with washing solution, the resultant material was analyzed using flow cytometry. '

Fig. 5a is a graph that cells treated with As₄0₆ and cells in untreated control group were labeled with FAM-DEVD-FMK using flow cytometry. In this graph, cells which catapase-3 was not activated were shown in a first section (Ml) of the X axis while cells which catapase-3 was activated were shown in a second section(M2). As shown Fig. 5a, when cells treated with 2.5μ M As₄O₆ for 18 hours, 19%) of the cells were stained in FAM-DEVD-FMK. From this result, it was shown that apoptosis induced by As₄0₆ in U937 cells was related to activation of catapase-3.

Additionally, in order to observe activation of catapase-3, Hoechst 33342 and PI were added in cell culture medium and, carried out reaction for 5 minutes to stain nucleus and then cells were observed with a fluorescent microscope. As shown in Fig. 5b, cells which catapase-3 was activated generated green fluorescence while dead cells stained with PI generated red fluorescence.

4) Western blotting analysis

The same procedure of Example 3 was performed on cell samples treated with 0, 0.5, 1, 2.5 and 5μ M As₄0₆ for 18 hours using catapase-3 activation detection kit. The resultant picture of Western blotting analysis was shown in Fig. 5c.

As shown in Fig. 5c, as concentration of Chunjisan treatment increases, catapase-3 was activated, thus degrading substrates to show a weak band.

Example 5: Experiment of identifying caspase-3 dependency

Using Z-DEVD-FMK, caspase-3 inhibitor the experiment was performed for identifying whether apoptosis induction by As₄0₆ was depending on inhibition of caspase-3 activation. The same procedure of Example 1-2) was performed on cell group treated with lμ M As₄O₆, other cell group treated with lμ M As₄O₆ and inhibiting apoptosis by Z-DEVD-FMK and normal control group. That is , the above cell groups were stained with annexin-FITC V/PI and the number of dead cells was measured using flow cytometry.

Additionally, the same procedure of Example 3 was performed on the above three experimental groups to measure mitochondria membrane potential depletion. The result was shown in Fig. 6.

As shown in Fig. 6, in comparison with control group, it was shown that 39.21%) of cells were dead and mitochondria membrane potential was depleted in cell group treated with As₄O₆. However, it was shown that apoptosis was not inhibited despite of treatment of 200μ M Z-DEVD-FMK, caspase-3 inhibitor. As a result, it was suggested that apoptosis induction of As₄0₆ was independent on caspase-3.

Example 6: Experiment for identifying relevancy with reactive oxygen 1) Inhibition of apoptosis by antioxidant In order to identify whether mitochondria membrane potential depletion during the process of inducing apoptosis by As₄0₆ in U937 cells is related to generation of reactive oxygen species in cells. N-acetyl-L-cysteine (NAC), antioxidant, was pretreated in the cells and As₄0₆ induced apoptosis was observed. After U937 cells (1 X 105) were pretreated with NAC 30 minutes before were treated with 2.5μ M As₄0₆, the same procedure was performed staining with annexin-FITC V/PI to measure the number of dead cells using flow cytometry. The procedure was three times repeated, and then the death ratio of cells was shown in Fig. 7a, wherein the number of dead cells in control group was 1.

The same procedure of Example 2 was performed on the treated cells to measure mitochondria membrane potential depletion. The degree of mitochondria membrane potential depletion was shown in Fig. 7b.

As shown in Figs. 7a and 7b, apoptosis induced by As₄O₆ was completely inhibited by 25mM NAC. From this result, it was suggested that generation, of reactive oxygen was related to As₄O₆ induced apoptosis.

2) Experiment for identifying generation of hydrogen peroxide

In order to identify whether apoptosis induced by As₄O₆ was related to generation of hydrogen peroxide, after treatment of As₄O₆ in U937 cells, generation of hydrogen peroxide was observed. 2',7'-dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR,

USA), material generating fluorescence by combining particularly with hydrogen peroxide, was used for observing generation of hydrogen peroxide in cells. 20μ M 2',7'-dichlorodihydrofluorescein diacetate was added in U937 cell groups treated with 2.5μ M As₄O_ό for 0.5, 1, 2, 3, 5 hours, respectively. After reaction for 30 minutes, the result was analyzed by flow cytometry. As shown in Fig. 7c, in comparision with control group, generation of hydrogen peroxide increased after 30 minutes. After 1 hour, generation of hydrogen peroxide increased by two times.

3) Inhibiting apoptosis by catalase

In order to identify whether generation of hydrogen peroxide was essential for apoptosis induction by As₄0₆, U 937 cells were pretreated with catalase that catalyzes the decomposition of hydrogen peroxide to oxygen and water and As₄0₆ induced apoptosis was investigated. U 937 cells (1 X 105) were pretreated with catalase, 30 minutes before

As₄O₆ was added. Then, the U937 cells were treated with 2.5μ M As₄0₆. The same procedure of Example 1-2) was performed on the cells staining with annexin- FITC V/PI to measure the number of dead cells using flow cytometry. The experiment was three times repeated, and then relative cell death ratio was shown in Fig. 7d, wherein the number of dead cells in control group was 1.

As a result, as shown in Fig. 7d, it was shown that apoptosis induced by As₄O₆ was completely inhibited in cells pretreated with 500U/ml catalase.

From the above-described Examples, it was shown that apoptosis induction by As₄O₆ was influenced by mitochondria membrane potential depletion caused by generation of hydrogen peroxide, Cytochrome c release in cytoplasm and activation of caspase-3.

Additionally, as shown in the above-described Examples, it was shown that hydrogen peroxide increased due to As₄0₆. As a result, it was suggested that apoptosis mechanism induced by As₄0₆ resulted from generation of hydrogen peroxide. Particularly, when cells were treated with NAC and catalase as antioxidant, apoptosis was completely inhibited. From this experimental result, it was suggested that apoptosis induced by As₄0₆ resulted from generation of hydrogen peroxide.

Accordingly, increased hydrogen peroxide by As 0₆ induces mitochondria membrane potential depletion and the membrane potential depletion induces Cytochrome c release in cytoplasm and successively actiyation of caspase-3, thereby inducing apoptosis in U937 cells.

INDUSTRIAL APPLICABILITY As discussed earlier, the tetraarsenic hexaoxide of the present invention may be used for induction apoptosis mechanism to prevent and treat many apoptosis related diseases. In addition, the tetraarsenic hexaoxide of the present invention is useful for blood cancers including leukemia as well as various solid tumors because it may effectively induce apoptosis even at its low concentration in U937, arsenic trioxide resistant leukemia cell.

Claims

What is claimed is:

1. An apoptosis inducer comprising As₄0₆.

2. The apoptosis inducer according to claim 1, wherein the apoptosis is induced by generating reactive oxygen.

3. A pharmaceutical composition for apoptosis induction comprising As₄O₆.

4. A blood cancer remedy comprising As₄O₆.

5. The blood cancer remedy according to claim 4, wherein the blood cancer is leukemia.