WO2003028001A2

WO2003028001A2 - Method and pharmaceutical composition for inducing cancer cell-specific apoptosis by combination with tnf family proteins and flavopiridol

Info

Publication number: WO2003028001A2
Application number: PCT/KR2002/001780
Authority: WO
Inventors: Shin-Wu Jeong; Dong-Myung Kim; Sung-Young Koo; Jin-Ho Lee; Chang-Yong Hong
Original assignee: Lg Life Sciences Ltd.
Priority date: 2001-09-24
Filing date: 2002-09-19
Publication date: 2003-04-03
Also published as: WO2003028001A8; WO2003028001A3; KR20030026069A; AU2002334433A1

Abstract

The present invention relates to a cancer cell- specific apoptosis inducing composition. The TNF (tumor necrosis factor; hereinafter, referred to as ' TNF ') family proteins and flavopiridol (also its salts) and the like are individually disadvantageous to provoke a serious side effect such as toxicity against an excessive dose, although they have excellent anti-cancer effects respectively. The composition of the present invention is prepared by combining the TNF family protein and flavopiridol, which reduces the toxicity or the side effect induced during the independent treatment at an high concentration, since the TNF family protein and flavopiridol are blended at an low concentration. Also, the composition has a specific action for cancer cells and a synergistic efficacy pharmaceutically.

Description

METHOD AND PHARMACEUTICAL COMPOSITION FOR INDUCING CANCER CELL-SPECIFIC APOPTOSIS BY COMBINATION WITH TNF FAMILY PROTEINS AND FLAVOPIRIDOL

TECHNICAL FIELD

The present invention relates to an apoptosis inducing method and pharmaceutical composition in which TNF (tumor necrosis factor; hereinafter, referred to as " TNF ") family proteins and flavopiridol are combined to induce the synergistic cell death (apoptosis) in human cancer cells.

BACKGROUND ART

Tumor necrosis factor, TNF is a cyto ine expressed in many types of cells and has various functions. It is generated in response to inflammation, infection, and other environmental stimuli. TNF induces a systemic or cellular response such as fever, tissue injury, tumor necrosis, in- appetency, secretion of another cytokines, cell growth, differentiation, apoptosis and the like. These responses are initiated by trimerization of TNF receptors, TNFRl (TNF receptor 1) and TNFR2 (TNF receptor 2) upon binding of TNF to the receptors. Most of the biological activities of TNF are mediated through TNFRl, but, to some extent, is also done via TNFR2. However, TNFR2 is supposed to be not involved in apoptosis by TNF.

An apoptosis by TNF is executed by activated caspases (cysteine-dependent aspartate-directed protease) via cascades of proteins bound to activated TNF receptors.

On the other hand, TNF is reported to activate NF-kB (nuclear factor-kappa B) . Various cell line studies show that NF-kB activation can inhibit the apoptosis by TNF in TNF-sensitive cells. On the contrary, down-regulation of NF-kB, in most cases, increases the sensitivity to apoptosis by TNF.

Precisely, lipopolysaccharides suppress an apoptosis of myeloid cells by TNF, through the activation of NF-kB, and Par-4 protein, which is expressed specifically in prostate carcinoma cells, increases an apoptosis by TNF through the inhibition of NF-kB activity. However, it has not been clarified yet how NF-kB controls an apoptosis. One possibility is that gene products expressed by NF-kB prevent the signal transduction system of an apoptosis. For instance, IAP (inhibitor of apoptosis) , TRAFl and TRAF2 are the genes controlled by NF-kB and seem to inhibit the activation of caspase-8. Therefore, it is expected to treat an infectious diseases caused by bacteria or virus, and cancers by combined application of NF-kB down-regulators and TNF. In addition to cancers, TNF is also known to be involved in other various diseases (arthritis, osteoporosis, asthma, etc.), in which NF- kB plays a role.

Despite a strong anti-cancer activity of TNF, it is restricted for. clinical applications as an anti- cancer agent due to severe systemic toxicity. Recently, combination of a high concentration of TNF, and melphalan, a kind of alkylating anti-cancer agent, show a substantial response in treating melanoma, sarcoma and hepatic cancer through the methods of an isolated limb perfusion or an isolated hepatic perfusion. TNF shows its anti-cancer activity not by its direct toxicity onto cancerous tissues but by indirect mechanisms such as damaging blood vessels in cancer tissues or activating immune reaction. Also it has been elucidated that micro and huge blood vessels of cancer are considerably damaged after operating an isolated limb perfusion with a combination of TNF, interferon-γ and melphalan. This positive effects lead to a study for reducing the toxicity of TNF to exploit a systemic administration at a therapeutic dose.

The therapeutic method for utilizing TNF-α for polycystic kidney disease has been specified in U.S. Patent No. 5,750,495.

TRAIL is a type II trans-membrane protein belonging to TNF family protein. Human TRAIL protein consists of 281 amino acids and has a homology of 28% with FasL (a cytokine associated with FAS receptor) and 23% with TNF, respectively. Till present, 5 types of Trail receptors have been reported in human, namely DR4, DR5 (also named with Apo-2, TRAIL-R2, TRICK2 or KILLER), DcRl (also named with TRID, TRAIL-R3, or LIT), DcR2 (also named with TRAIL-R4 or TRUNDD) and TR1. Binding of TRAIL to DR4 or DR5 results in a trimerization of the receptors and thus, induces apoptosis by activating caspases in a terminus of signal transduction pathways similar to that of TNF. But, TRAIL do not show cell toxicity in normal cells, which is different from that of TNF, and FasL and act as a anti-cancer agent without showing the systemic toxicity when injected to a mouse. Therefore, TRAIL is a promising anticancer agent. The cell cycle of eukaryotic cell is progressed through the cascade of activations of cyclin-dependent kinases (CDKs) . Some small molecular weight tumor suppressor proteins such as p21, p27, pl5, pl6 control the kinase activities. In many human cancer cells, these proteins are often observed to be mutated or controlled abnormally.

These results have suggested that synthetic compounds inhibiting functions of CDK' s may prevent a cell division and treat cancers by inhibiting a cell cycle, and among these, flavopiridol is a major CDK inhibitor under a clinical trial. Flavopiridol especially controls activities of CDK2 and CDK4 to suppress an excessive cell proliferation by stopping a cell growth at Gl phase, and eventually treats cancer (Carson et al., 1996, Cancer Res . 56: 2973-2978).

U.S. Patent No. 6,087,366, has disclosed that this flavopiridol or its salts is used for the therapeutic method for Alzheimer's disease or Parkinson' s disease by controlling a cell cycle of a nerve cell. Besides, U.S. Patent No. 6,225,473, has disclosed the process for preparing flavopiridol. Experiments on the xenograft assay of human cancer cells in mouse have proved that flavopiridol inhibit the proliferation of cancer cells effectively. However, it has showed many side effects in human clinical trials. As mentioned above, TNF, TRAIL, flavopiridol and the like, in spite of their competent anticancer activity, have several side effects clinically due to high concentration of individual treatment.

DISCLOSURE OF INVENTION

Therefore, in order to settle the above disadvantages, the object of the present invention is to provide a pharmaceutical composition and a method for preparing the same in which the TNF family protein and flavopiridol are combined to induce the synergistic cell death (apoptosis) of cancer cells.

In order to attain the object described above, the present invention provides a method for inducing cancer cell-specific apoptosis by co-treatment of TNF family proteins and flavoiridol including its salts. The present invention provides a pharmaceutical composition for inducing cancer cell-specific apoptosis containing TNF family proteins and flavopiridol.

The TNF family protein is more than one selected from the group consisting of TNF- (tumor necrosis factor), TRAIL (TNF-related apoptosis inducing ligand) and protein variants containing a N-terminus or a C- terminus of the TNF- α OR TRAIL.

The concentration of the TNF family protein is at the range of 1.0 ~ 2,000 Pg/ml, and the concentration of the flavopiridol is at the range of 1.0 ~ 1,000

Ug/ml.

The cancer cell specific apoptosis inducing compositions used in the present invention has increased efficacy more remarkably than that of the independent use, by using the composition of TNF family protein and flavopiridol.

The TNF family proteins used in the present invention can be TNF-α, TRAIL (TNF-related apoptosis inducing ligand) and any other proteins with a similar property.

The flavopiridol is not limited into the compound having a formula depicted in the present invention and also includes these salts or any other substance with a similar property.

In order to investigate and support the effects of the present invention, TNF-α, TRAIL, flavopyridol (NSC649890, L86-8275, (-) cis-5, 7-dihydroxy-2- (2- chlorophenyl) -8- [4- (3-hydroxy-l-methyl) -piperidyl] -4H- l-benzopyran-4-one) and so on are treated separately or in a combined mode into various kinds of cells such as human cancer cells and the like, and then the cytotoxicity is estimated by analyzing FACScan or DNA fragments .

In order to elucidate a lot of biological changes in these drug treated cells, Western blot and transient transfection assays are performed. To elevate the apoptosis effect, the compositions of the present invention can be made of a conventionally and pharmaceutically acceptable additive agent. Preferably, the TNF family protein can be contained at the range of 1.0 ~ 2,000 μg/ml and flavopiridol can be contained at the range of 1.0 ~ 1,000 μg/ml. BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings.

FIG. 1 represents a synergistic apoptosis by treating TNF-α and flavopiridol coincidently on various human cancer cell lines.

FIG. 2 represents the time-dependency for apoptosis by the combined treatment of TNF and flavopiridol in a human lung cancer cell line, A549.

FIG. 3 represents the comparison of apoptosis induced by combined (TNF-α plus flavopiridol) and single (flavopiridol only) treatments.

FIG. 4 represents the drug concentration effects for apoptosis, compared with the results of combined treatments of both TNF-α and flavopiridol.

FIG. 5 represents SDS-PAGE gel electrophoresis patterns of some intracellular proteins after the combined treatment of both TNF-α and flavopiridol . FIG. 6 represents the effects of TNF-α and flavopiridol on NF-kB transcriptional activities.

FIG. 7 represents an agarose gel electrophoresis pattern that shows DNA laddering in combined treatment of both TNF and flavopiridol.

FIG. 8 represents non-toxic effects of combine treatment of TNF-α and flavopiridol on a normal cell line, rat2.

Best Mode for Carrying Out the Invention

Practical and presently preferred embodiments of the present invention are illustrated as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

<Example 1> Synergistic apoptosis induced by a combination of TNF-α and flavopiridol or TRAIL and flavopiridol in various cell lines of human cancers

Human cancer cell lines were cultured in 5% C0₂ incubator with RPMI1640 medium containing 5% of FBS (fetal bovine serum) (also containing 1,000 units/ml penicillin G, 100 μg/ml of streptomycin G, 0.25 μg/ml amphotericin B, 2mM of gluta ine) at 37°C. The cancer cell lines used were as like rectal cancer cell lines HCT-116, bladder cancer cell lines T24, rectal cancer cell lines HCT15, and lung cancer cell lines A549. FACScan (flow cytometric analysis) was performed to measure an apoptosis generated in cancer cells. Detailed experimental procedures were as follows.

First, 5 x 10⁵ cells of the cancer lines were seeded to a culture dish with 60 mm of a diameter and cultured in 5 ml of the above cell medium for a day. Then, flavopiridol (0.5μM) was combined with TNF-α (purchased from Boehringer Mannheim company, 10 ng/ml) or TRAIL (purchased from Serotec company, 50 ng/ml) and treated simultaneously. After treating for a marked time, the medium in an upper layer was gathered to 15 ml of a falcon tube (purchased from Becton Dickinson company) . Sticking cells were washed twice with 5 ml of PBS buffer, and separated after being treated with 0.5 ml of 0.15% trypsin, and then mixed with the stored medium in an upper layer. After that, the tube was centrifuged at 200 x g (gravity) for five minutes to precipitate the cells. After eliminating the supernatant, the precipitated cells were suspended with 300 μl of PBS buffer, and carefully added with 5 ml of pre-cooled 75% ethanol at 4 °C during being vortexed, and fixed after all. The fixed cells were left for at least 3 hours and then centrifuged again to eliminate ethanol. PBS buffer containing RNase (purchased from Sigma company) with 0.1 mg/ml. concentration was added to the cell until 1 x 10⁶/ml of a cell density and propidium iodide (1 mg/ml, distilled water) was also added to stain the cell until (reaching) a final concentration of 50 μg/ml in light-tight condition.

Next, 10,000 cells among the stained cells were analyzed through FACScan by using FACSort (purchased from Becton Dickinson company) with 488 nm of excitation wavelength and 588 nm of emission wavelength. Modifit software (purchased from Variety software house company located in Maine state, Topsham) was exploited to analyze numerical value of a histogram.

In case TNF-α and flavopiridol or TRAIL and flavopiridol were combined, a considerable synergistic apoptosis was induced as depicted in following table 1. This result was also obtained from experiments with several kinds of cells as presented in FIG. 1 (See FIG. 1: after adding lOng/ml of TNF-α and 0.5μM of flavopiridol, the cells were cultured for 24 hours. C; control, T; 10 ng/ml of TNF-α, F.P; flavopiridol (0.5μM), +; lOng/ml of TNF-α and 0.5μM of flavopiridol are simultaneously treated. )

<Table 1> synergistic apoptosis effects in A549, human small lung cancer cell lines, by combined treatment of TNF-α or TRAIL and flavopiridol, a CDK inhibitor

<Example 2> Effects of time-dependency for apoptosis by the combined treatment of TNF and flavopiridol in a human small lung cancer cell line, A549.

After culturing A549 cell lines with the same

^•method in Example 1, lOng/ml of TNF-α and 0.5μM of flavopiridol were co-treated and cultured for 0, 3, 6,

12, 24 hours separately. Then, FACScan was performed with the same method described in Example 1.

Under the same conditions described in the following Table 2 and FIG. 2, an apoptosis was increased gradually with time-dependency. (See FIG. 2)

Increase of synergistic apoptosis of A549 cell lines with the time-dependency of co-treatment of TNF-α and flavopiridol .

<Comparative Example 1> Effect of treatment sequence of TNF-α and flavopiridol on apoptosis

A549 cell lines were cultured through the same method described in Example 1. lOng/ml of TNF-α was treated for 6 hours, washed twice with 5ml of PBS, and then 0.5μM of flavopiridol was treated for another 6 hours. Otherwise, TNF-α and flavopiridol were treated with opposite sequence for 6 hours respectively or co- treated for 6 hours or 12 hours respectively. After that, FACScan was performed with the same method described in Example 1 after fixation step.

As depicted in the following Table 3, a considerable apoptosis was induced only when TNF-α and flavopiridol are co-treated. As a result, a synergistic apoptosis can be induced when cells are exposed to both agents simultaneously.

Effects of treatment sequence of TNF-α and flavopiridol on apoptosis.

<Comparative Example 2> Effects of treatment sequence of TNF-α and flavopiridol on apoptosis.

A549 cell lines were cultured with the same method described in Example 1. lOng/ml of TNF-α was treated for 3 hours. After eliminating the media, lOng/ml of TNF-α and 0.5μM of flavopiridol were additionally co-treated for another 6 hours. Otherwise, 0.5μM of flavopiridol was treated for 3 hours. After eliminating the media, lOng/ml of TNF-α and 0.5μM of flavopiridol were additionally co-treated for another 6 hours .

And also, either 10ng/ml of TNF-α or 0.5μM of flavopiridol was treated for 9 hours individually, and then FACScan was performed after fixing the cells with the same method described in Example 1.

As shown in the following Table 4, an apoptosis does not appear in the group in which TNF-α was treated for 3 hours before the co-treatment, while a remarkable apoptosis appears in the group in which flavopiridol was treated first before the co-treatment.

Effects of treatment sequence of TNF-α and falvopiridol on apoptosis

<Example 3> Comparative analysis of apoptotic potency by co-treatment of TNF-α and flavopiridol

After A549, human small lung cancer cell lines, was cultured with the same method described in Example 1, TNF-α and flavopiridol were treated individually or simultaneously for 6 hours at each concentration. Then, the amount of apoptosis was compared by FACScan. In case of individual treatment, both 50μM of flavopiridol and 50ng/ml of TNF-α did not show any significant apoptosis (See FIG. 3, FIG. 4(A)). On the contrary, a remarkable apoptosis was induced when both agents were co-treated in low concentrations. Additionally, the quantity of apoptosis was shown to be dependent on the concentration of flavopiridol in the presence of TNF- α (See FIG. 4(B)).

<Example 4> Analysis on functions of caspase for inducing apoptosis by co-treatment of TNF-α and flavopiridol

After culturing A549 with the same method described in Example 1, peptide inhibitors (purchased from Alexis biochemical company) of various caspases were treated at various concentrations. After 30 minutes, TNF-α and flavopiridol were co-treated and cultured for 24 hours. Next, cells were fixed with the same method described in Example 1, and then apoptotic portions were analysized by FACScan.

As described in the following Table 5, caspase inhibitors inhibited apoptosis with concentration- dependency. Especially, Z-YVAD-FMK was outstanding in inhibiting apoptosis. As a result, it is elucidated that various activated caspases play a role in inducing an apoptosis by co-treatment of TNF-α and flavopiridol . <Table 5> Analysis on the functional role of caspases for synergistic apoptosis in A549, a human small lung cancer cell line, when TNF-α and flavopiridol were co- treated: lOng/ml of TNF-α and 0.5μM of flavopiridol were treated. Caspase peptide inhibitors were treated at each concentration of 3, 10, and 30 μM 30 minutes before the co-treatment of TNF-α and flavopiridol.

<Example 5> Measurement of quantitative changes of intracellular proteins by co-treatment of TNF-α and flavopiridol

4 x 10⁶ A549 cells, human small lung cancer cell line, were cultured in a 100mm tissue culture dish with the above culture medium for a day, and TNF-α and flavopiridol were treated separately or simultaneously for 24 hours with the concentration. Then, cells were carefully washed twice with 10 ml of PBS, and 1ml of PBS containing protease inhibitor cocktail (purchased from Roche company, complete™-mini) was poured to each culture dish. After that, cells were collected and destroyed with an ultrasonicator . The obtained sample was centrifuged at 12000x rpm for 20 minutes with a micro-centrifuge, the supernatant was collected, and proteins were quantified with Bradford dye reagent (purchased from Bio-Rad company) . Thereafter, 40μg of proteins were loaded on a SDS-PAGE gel, transferred to nitrocellulose membrane (purchased from Bio-Rad company) , and quantitative change of each protein was measured by exploiting primary antibody, which is specific for a target protein, secondary antibody

(purchased from Amersham or Bio-Rad company) , which is conjugated with HRP (horseradish peroxidase) , and ECL chemiluminescence reagent (purchased from Amersham company) .

Caspase-3 and caspase-8 were shown to be activated by co-treatment of TNF-α and falvopiridol . Moreover, Rb, PARP and Lamin B protein, which are substrates of caspases were shown to be degraded (See FIG. 5 : C; control, T; lOng/ml of TNF-α, F.P; flavopiridol with the marked concentration, +; co- treatment of lOng/ml of TNF-α and 0.5μM of flavopiridol) .

<Example 6> Measurement of changes in transcriptional activity of NF-kB by co-treatment of TNF-α and flavopiridol

Each of 4 x 10⁶ A549, human small lung cancer cell line, was cultured in two lOOmm tissue culture dishes for 24 hours in the same culture medium described in Example 1. Then, each culture dish was washed with 10ml of RPMI medium without FBS (fetal bovine serum) , and pNF-kB-Luciferase plasmid (purchased from Stratagene company, catalog No. 219078) was transfected to one culture dish by using lipofectAMINE PLUS™ eukaryotic cell transfection system (purchased from Gibco-BRL company) in accordance with a manufacturer' s method, while a control group except pNF-kB-Luciferase plasmid was treated to the other culture dish. pNF-kB-Luciferase plasmid contains 5x NF-kB responsible elements in promoter region upstream of luciferase-coding sequence, so that the production of luciferase proteins depends on the NF-kB activity. A detailed description is as follows .

9 μg of plasmid DNA was mixed with 54 μl of plus reagent (purchased from Gibco-BRL company) in a polystyrene tube with a round bottom in advance, and finally reached 900 μl with adding RPMI 1640 medium without FBS, while TE (10 μM Tris, pH 8.0, 1 mM EDTA) solution with the same volume was mixed in a control group. And in the other tube containing 36 μl of lipofectAMINE reagent (purchased from Gibco-BRL company) , RPMI 1640 medium without FBS was added until a final volume of 900 μl . After leaving at room temperature for 15 minutes, each soultion was mixed, and then left again at room temperature for another 15 minutes. Thereafter, this mixture was transfected by dropping to A549 culture dish which is exchanged to 7.8 ml of RPMI 1640 medium without FBS. After culturing in a cell culture incubator with 5% C0₂ at 37 °C for 5 hours, a medium was exchanged again to RPMI

1640 medium with 5% of FBS, and the solution was cultured for another 1 hour. Then, each culture dish was treated with 1 ml of 0.1% trypsin solution

(purchased from Gibco-BRL company) to separate the cells from dishes. Then the detached cells were transferred to 30 ml of RPMI1640 medium containing 5% FBS , mixed thoroughly and divided to 12-well dish by 2.5 ml for each well. After culturing in an incubator for 16 hours, final 10 ng/ml of TNF-α and 0.5 μM of flavopiridol were treated separately or simultaneously in duplicate and cultured again for 6 hours.

Next, Luciferase activity in cell extracts was measured by using luciferase assay system (purchased from Promega company) according to manufacturer's instructions. In this case, intensity of a light (Luciferase activity) was measured with Lumat model LB953 Luminometer (purchased from Berthold company in Germany) . A protein quantity in cell extract was measured by BCA (bicarbonate assay) method using BSA (bovine serum albumin) as a standard, and this value was used to assure the luciferase activity to represent the same protein quantity.

In A549 cell, TNF increased luciferase activity, while flavopiridol did not affect it. TNF-α combined with flavopiridol did not increase luciferase activity(See FIG. 6).

<Ξxample 7> Observation of DNA laddering by co- treatment of TNF and flavopiridol

A549 cells (lxlO⁶ cells) was plated to a 60mm culture dish and cultured for a day. Flavopiridol and

TNF-α were treated separately or simultaneously to the cells with a marked concentration for 24 hours and cells were collected to separate DNA through the following process. Cells floating on the medium and sticking to a bottom of the medium were collected, washed with 1ml of PBS (phosphate buffered saline) , and centrifuged with a micro-centrifuge at 5000 rpm for 5 minutes to attain a cell pellet. After discarding the supernatant, lysis buffer (1% NP-4Q, 20mM EDTA, 50mM

Tris-HCl, pH 7.5) was poured and mixed carefully with pippeting to lysis the cells. The cell lysate was centrifuged at 5000 rpm for 5 minutes and a supernatant was transferred to a new tube. NaCl and SDS were added to the supernatant until a final concentration of 0.2M and 0.5% respectively, and PCI solution (phenol: Choloroform: isoamylalchol = 25:24:1) with the same volume with the supernatant was added and vortexed. The prepared solution was centrifuged at 14,000 rpm for 2~5 minutes, a supernatant was collected, and phenol extraction was performed repeatedly twice. Ethanol was added to the supernatant to attain the DNA pellet, and the DNA pellet was dissolved to lOμl of water and reacted with RNase A at 37 °c for 10 minutes to eliminate RNA. Fresh proteinase K was treated at 37 °c for 10 minutes to eliminate proteins in the solution. Thereafter, DNA was precipitated with a phenol extraction and an ethanol precipitation (sedimentation) technique, analyzed through electrophoresis in 1.5% of agarose gel, and confirmed under UV with dying with EtBr. As described in FIG. 7, DNA laddering which is a characteristic of an apoptosis was remarkably found in cells treated with flavopiridol and TNF-α simultaneously (See FIG. 7 : M; 1 Kb DNA marker, C; control(0.1% DMSO) , D; 1 M doxorubicum, T; lOng/ml of TNF-α treated, F.P: flavopiridol treated with a marked concentration, +; treating 10ng/ml of TNF-α and 0.5μM of flavopiridol) . <Ξxample 8> Confirmation of non-cytotoxic effect on rat2 , a rat' s normal fibroblast cell lines in treating TNF-α and flavopiridol simultaneously

5 x 10⁵ rat2 cells, a rat's cell lines, were inoculated to a culture dish of 60mm and cultured for 24 hours with the same condition described in Example 1. After that, lOng/ml of TNF-α and 0.5μM of flavopiridol were separately or simultaneously treated and cultured for 6 hours. Thereafter, cells were separated with the same method described in Example 1 and analyzed with FACScan.

As shown in FIG. 8, no apoptosis was confirmed in normal cell lines, rat2, though TNF-α and flavopiridol are co-treated, which is different from a case in cancer cell lines. An apoptosis is observed just in co-treatment of TNF-α and flavopiridol to cancer cells and this proves an anticancer characteristic (See FIG. 8)

INDUSTRIAL APPLICABILITY

As demonstrated clearly and confirmed above, the present invention provides an apoptosis inducing composition in which the TNF family protein and flavopiridol are combined in a proper ratio, and can increase apoptosis specific for cancer cells synergistically. Therefore, the composition of the present invention can be utilized usefully and widely.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention, and equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

We claim:

1. A Method for inducing cancer cell-specific apoptosis by co-treatment of TNF family proteins and flavopiridol including its salts.

2. The Method for inducing cancer cell-specific apoptosis according to claim 1, wherein said TNF family protein is more than one selected from the group consisting of TNF-α (tumor necrosis factor) , TRAIL (TNF-related apoptosis inducing ligand) and protein variants containing a N-terminus or a C- terminus of said TNF-α or TRAIL.

3. A Pharmaceutical composition for inducing cancer cell-specific apoptosis containing TNF family proteins and flavopiridol .

4. The Pharmaceutical composition for inducing cancer cell-specific apoptosis according to claim 3, wherein said TNF family protein is more than one selected from the group consisting of TNF-α (tumor necrosis factor) , TRAIL (TNF-related apoptosis inducing ligand) and protein variants containing a N- terminus or a C-terminus of said TNF-α or TRAIL.

5. The Pharmaceutical composition for inducing cancer cell-specific apoptosis according to claim 3, wherein a concentration of said TNF family protein is at the range of 1.0 ~ 2,000 μg/ml, and a concentration of said flavopiridol is at the range of 1.0 ~ 1,000 μg/ml .