KR102169203B1 - Combination therapy of cdk7 inhibitor bs-181 and death receptor ligand trail for prevention or treatment of cancer - Google Patents

Abstract

The present invention is directed to the concurrent administration of a CDK7 inhibitor (cyclin-dependant kinase 7 inhibitor), the BS-181 compound of the following formula (I) and a death receptor ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). It relates to a medical use for the prevention or treatment of cancer caused by. The combined treatment of BS-181 and rTRAIL as a pharmaceutical use for the prevention or treatment of cancer of the present invention has a synergistic effect of remarkably increasing the cytotoxicity of BS-181 against malignant tumor cells (Jurkat A3, U937 and HeLa). Although shown, there was no effect on normal human T cells. In particular, the combined treatment of rTRAIL (1-4 ng/ml) and BS-181 enhances the induction of exogenous apoptosis, so that the IC ₅₀ value of BS-181 for Jurkat A3 is not toxic to normal T cells. It has been shown that the concentration can be lowered. These results show that the combination of BS-181 and rTRAIL can be usefully applied to the treatment of cancer, especially T-ALL.

Description

COMBINATION THERAPY OF CDK7 INHIBITOR BS-181 AND DEATH RECEPTOR LIGAND TRAIL FOR PREVENTION OR TREATMENT OF CANCER}

The present invention is directed to the concurrent administration of a CDK7 inhibitor (cyclin-dependant kinase 7 inhibitor), the BS-181 compound of the following formula (I) and a death receptor ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). It relates to a medical use for the prevention or treatment of cancer caused by.

Human T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive tumor produced by malignant transformation of T cell lineage lymphocytes, accounting for about 20% of all acute lymphoblastic leukemia [1]. ,2]. Although the overall survival rate of T-ALL in children and adults has improved due to the recent advances in risk-adapted chemotherapy, the prognosis is good because resistance to chemotherapy and early recurrence may occur in T-ALL in high-risk groups. And the survival rate is low [3,4]. In order to improve the overall survival rate in chemotherapy treatment for T-ALL and other high-risk leukemias, there is high demand for new anticancer drugs that can minimize drug resistance and side effects [5].

Cell cycle progression is positively regulated by a family of cyclin-dependent kinases (CDKs). The CDK family includes three interstitial CDKs (CDK2, CDK4, CDK6), one mitotic CDK (CDK1), regulatory CDK (CDK7, a catalytic subunit of the CDK-activating kinase complex), and two transcriptional CDKs (CDK8, CDK9) is included [5-8]. With respect to uncontrolled proliferation of tumor cells, the catalytic activity of CDKs is often increased due to genetic and epigenetic changes in their regulatory proteins such as CDKs, CDK inhibitors and cyclins [9-13]. ]. Therefore, CDK is emerging as an attractive target for the development of anticancer and chemotherapy drugs [8,9,14].

Compared to other CDK families, the role of CDK7 in the cell cycle is that it forms a CDK-activating kinase (CAK) complex that is responsible for phosphorylation of CDK1, CDK2, and CDK4 together with cyclin H and MAT1, and that the CAK complex forms the transcription factor TFIIH. It exists as part and is unique in that it phosphorylates the C-terminal domain of the largest subunit of RNA pol II for initiation of transcription of the RNA pol II-directing gene. In addition, CAK or TFIIH-related CAK is known to regulate their activity by phosphorylating several other transcription factors [15-17]. These previous studies suggest that CDK7 may be an important therapeutic target for tumors as an essential regulator of cell cycle progression and transcription.

Recently, BS-181, a pyrazolo [1,5-a] pyrimidine-inducing compound, is known as a specific CDK7 inhibitor (CDK7 inhibitor), and has a cytotoxic effect on solid tumors including gastric cancer, G ₁ cell cycle arrest and mitochondrial- There are reports that it is associated with dependent apoptosis [18,19]. However, in order to clarify the anticancer activity of BS-181, further investigation is needed on the correlation between cell cycle arrest and apoptosis. Moreover, the fact that the exogenous apoptosis pathway is involved in the induction of apoptosis by BS-181 is not known at all, and whether the anticancer activity of BS-181 observed in solid cancer cells can also appear in T-ALL and other leukemia cells. It is also unclear whether or not.

The present invention was devised to solve the problems of the prior art as described above, and the problem to be solved in the present invention is for the prevention or treatment of cancer comprising a compound of the following formula (I), which is a CDK7 inhibitor, and the death receptor ligand TRAIL It is intended to provide a pharmaceutical composition.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof, and TRAIL as an active ingredient:

(Ⅰ)

It is preferable that the TRAIL is a recombinant human TRAIL.

The cancer is gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer. ), head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer , Colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer ), parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchogenic cancer, and bone marrow tumor ) It is preferably selected from the group consisting of.

It is preferred that the cancer is acute T lymphocytic leukemia.

In addition, the present invention provides a functional food for preventing or improving cancer, including the compound of Formula (I) or a pharmaceutically acceptable salt thereof, and TRAIL.

It is preferable that the TRAIL is a recombinant human TRAIL.

The cancer is stomach cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer , Cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, it is preferably selected from the group consisting of bronchial cancer and bone marrow cancer.

It is preferred that the cancer is acute T lymphocytic leukemia.

The present invention also provides a in vitro popto cis induced promotion how the cell of which comprises on the (in vitro), handle to the cells a compound or a pharmaceutically acceptable salt thereof of formula (Ⅰ), and TRAIL .

It is preferable that the TRAIL is a recombinant human TRAIL.

It is preferable that the cell is a T cell.

The combined treatment of BS-181 and rTRAIL as a pharmaceutical use for the prevention or treatment of cancer of the present invention has a synergistic effect of remarkably increasing the cytotoxicity of BS-181 against malignant tumor cells (Jurkat A3, U937 and HeLa). Although shown, there was no effect on normal human T cells. In particular, the combined treatment of rTRAIL (1-4 ng/ml) and BS-181 enhances the induction of exogenous apoptosis, so that the IC ₅₀ value of BS-181 for Jurkat A3 is not toxic to normal T cells. It has been shown that the concentration can be lowered. These results show that the combination of BS-181 and rTRAIL can be usefully applied to the treatment of cancer, especially T-ALL.

FIG. 1 shows CDK7 in Jurkat T cell clones (JT/Neo cells and JT/BCL-2 cells) transfected with empty vector (JT/Neo) or BCL-2 expression vector (JT/BCL-2). This is a result showing a decrease in cell viability induced by treatment with the inhibitor BS-181, a change in cell cycle distribution, and apoptosis due to apoptosis.
FIG. 2 shows the loss of ΔΨm and BAK activation induced by BS-181 in JT/Neo and JT/BCL-2 cells, apoptosis regulators [BCL-2, BCL-XL, MCL-1, BAK , BID, BIM isoforms (BIMEL and BIML), XIAP, caspase-9, caspase-8, caspase-3, PARP] and changes in exogenous apoptosis regulators (DR5 and TRAIL), and cell surface TRAIL and secretion TRAIL This is a result showing an increase in
3 is a result showing the decrease in Rb phosphorylation induced by CDKs and Cyclins levels, and CDKs in the JT / Neo cells and JT / BCL-2 cells treated with BS-181.
4 is a result showing the effect of hydroxyurea (HU) or paclitaxel (PTX) on the accumulation and Δψm loss of apoptotic sub-G ₁ cells induced by BS-181 treatment in JT/Neo cells.
Figure 5. FADD- and caspase-8-positive wild type Jurkat T cells (clone A3), FADD-deficient Jurkat T cells (clone I2.1) and caspase-8-deficient Jurkat T cells (clone I9.2). The differential effect of BS-181 on the cell viability, cell cycle distribution, loss of ΔΨm, and expression levels of DR5, TRAIL, BID, FADD, caspase-9, caspase-8, caspase-3, PARP, and GAPDH This is the result showing
Figure 6 shows FADD- and caspase-8-positive wild type Jurkat T cells (clone A3), FADD-deficient Jurkat T cells (clone I2.1) and caspase-8-deficient Jurkat T cells (clone I9.2). It shows the synergistic effect of rTRAIL on the cytotoxicity of BS-181 against Korea.
Fig. 7 shows the results of investigation of the effects of administration of BS-181 or recombinant TRAIL alone, and combined treatment with BS-181 and rTRAIL on A3 cells on cell cycle distribution, loss of Δ Ψm, and induction of apoptosis.
8 is a result showing the effect of BS-181 on the proliferation of IL-2-dependent proliferation of non-activated peripheral blood T cells and PHA-activated T cells of normal humans and Jurkat T cells (E6.1).

Hereinafter, the present invention will be described in detail.

In the last 20 years, intensive research has been conducted on CDK inhibitors as new anticancer drugs. Although the first-generation CDK inhibitors with broad specificity showed low efficacy in clinical trials, second-generation CDK inhibitors targeting a narrow range of CDK are recognized as promising chemotherapeutic agents as clinical results, and recently, specificity for CDK4/6. Inhibitors such as palbociclib and ribociclib have been approved by the FDA as chemotherapy treatments for breast cancer [36-38]. In addition, with an effort to extend the target of the CDK inhibitor to non-canonical CDKs (non-canonical CDKs) such as CDK7, CDK8 and CDK9, an attempt to develop a third generation CDK inhibitor having a more specific efficacy against the target CDK has been attempted. Further progress is being made. BS-181 was confirmed to selectively inhibit the activity of CDK7 (catalytic subunit of CAK) in vitro, even at low nanomolar concentrations, and was developed as a third-generation CDK inhibitor using structure-based computer-assisted drug design technology. [18].

In the present invention, it was first discovered that the anticancer activity of BS-181 is due to the induction of exogenous apoptosis in Jurkat T cells (JT/Neo) through upregulation of TRAIL and DR5. BS-181 induces G ₁ -arrest and apoptosis due to downregulation of G ₁ cyclin (cyclin D1) and anti-apoptotic proteins (MCL-1 and XIAP) in breast cancer cells [18] and gastric cancer cells It has been reported that G ₁ -arrest and apoptosis are caused by downregulation of cyclin D1 and XIAP in [19]. In contrast, in Jurkat T cells, G ₁ -arrest following BS-181 treatment was observed in JT/BCL-2 cells in which apoptosis induced by BS-181 is inhibited by overexpression of BCL-2. , It was not observed in JT/Neo cells. These results suggest that the induction of apoptosis according to BS-181 treatment can be caused preferentially in G ₁ -cells, so that the late G ₁ -stopper, HU [32,33] or the pre-medium-stopper, PTX [39 ] On the induction of apoptosis by BS-181 was investigated in JT/Neo cells. As a result, it was found that the apoptosis induced by BS-181 was significantly increased by HU, but somewhat suppressed by PTX. In addition, JT/Neo cells treated with 15 μM BS-181 were stained with FITC-Annexin V and DAPI, then fixed with paraformaldehyde, treated with RNase, and PI stained. At this time, FITC-Annexin was not stained with DAPI. As a result of investigating the cell cycle distribution according to PI staining of early apoptotic cells stained only with V by flow cytometry, the initial apoptotic cells stained with FITC-Annexin V were 40.1% of apoptotic sub-G ₁ cells and 35.4. % Of G ₁ cells, these results indicate that the apoptosis response induced by BS-181 treatment in JT/Neo cells, cell cycle progression fails to enter S phase due to inhibition of CDKs-mediated Rb phosphorylation. This confirms that it is preferentially induced in cells stopped in late G ₁ phase.

On the other hand, the levels of CDK7 and cyclin H were not significantly changed in JT/Neo and JT/BCL-2 cells treated with BS-181, but G ₁ -cyclins (cyclin D1, cyclin D3) and G ₁ -CDK (CDK4, CDK2), and the levels of M-CDK and M-cyclin, CDK1 and cyclin B1, were significantly decreased. In particular, CDK7-induced phosphorylation of Rb by CDK1 (Thr-161) and CDK2 (Thr-160) and CDK1/2- and CDK4/6 was more clearly reduced in JT/BCL-2 cells than in JT/Neo cells. Confirmed. Since Rb's ability to block cell cycle progression at the G ₁ -checkpoint is lost when overphosphorylated by CDKs, it is known to allow G ₁ /S conversion [40], so these findings are based on JT/BCL-2. The G ₁ -arrest following BS-181 treatment observed in cells confirms that Rb hyperphosphorylation by CDKs is due to a failed result in the presence of BS-181 acting as a CDK7 inhibitor.

In the present invention, the contribution of exogenous cell death in BS-181 cytotoxicity to Jurkat T cells is not only western blotting showing an increase in DR5 levels after BS-181 treatment in JT/Neo cells, but also TRAIL elevation on the cell surface and cell It was first predicted by surface immunofluorescence staining and ELISA analysis data indicating an increase in TRAIL. These predictions indicate that FADD-deficient Jurkat I2.1 cells and caspase-8-deficient Jurkat I9.2 cells [34,41], both of which cannot induce exogenous apoptosis, were resistant to the cytotoxic effects of 10 μM BS-181. On the other hand, wild-type Jurkat A3 cells were confirmed by the study results showing a cell survival rate of 67%. Both I2.1 and I9.2 cells treated with 10 μM BS-181 failed to induce apoptosis, but treatment with 15 μM BS-181 successfully produced intrinsic apoptosis through the BAK-mediated mitochondrial pathway, At this time, the observed sub-G ₁ ratio increased to ~28% of the A3 cell.

In conclusion, treatment of human T-ALL cell line Jurkat cells with a pharmacological concentration of BS-181, a CDK7 inhibitor, showed anti-cancer activity (IC ₅₀ = 14.5 μM) mainly through exogenous TRAIL/DR5 upregulation-mediated epoptosis induction. Show. Although, by BS-181 treatment, the endogenous BAK-dependent mitochondrial-mediated apoptosis pathway can be initiated, apart from the induction of exogenous apoptosis, apoptosis by exogenous apoptosis rather than endogenous apoptosis. It was confirmed that BS-181 is a major mechanism of cytotoxicity against Jurkat T cells.

On the other hand, the combined treatment of BS-181 and rTRAIL showed a synergistic effect of remarkably increasing the cytotoxicity of BS-181 against malignant tumor cells (Jurkat A3, U937 and HeLa), but against normal human peripheral blood T cells. There was no synergy effect. In particular, the combined treatment of rTRAIL (1-4 ng/ml) and BS-181 enhances the induction of exogenous apoptosis, so that the IC ₅₀ value of BS-181 for Jurkat A3 is not toxic to normal T cells. It has been shown that the concentration can be lowered.

These results show that the combination of BS-181 and rTRAIL can be usefully applied to the treatment of cancer, especially T-ALL.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the compound of Formula (I) or a pharmaceutically acceptable salt thereof, and TRAIL as an active ingredient:

(Ⅰ)

The compound of formula (I) may exist in the form of a base-addition salt or an acid-addition salt. Such addition salts are included in part of the present invention. Such salts are advantageously prepared with pharmaceutically acceptable acids, but salts of other acids useful, for example, for purifying or isolating compounds of formula (I) are also included in the present invention. The acids are, for example, picric acid, oxalic acid or optically active acids such as tartaric acid, dibenzoyltartaric acid, mandelic acid or camphorsulfonic acid, and physiologically acceptable salts such as hydrochloride, hydrobromide, sulfate, hydrogen. It may be an acid forming sulfate, dihydrogen phosphate, maleate, fumarate, 2-naphthalenesulfonate or para-toluenesulfonate. For physiologically acceptable salts, reference may be made to Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002).

The TRAIL is preferably a recombinant human TRAIL, for example, as TRAIL of human origin.

The cancer is stomach cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer , Cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer and bone marrow cancer, preferably selected from the group consisting of, and more preferably acute T lymphocytic leukemia. Do.

The pharmaceutical composition of the present invention is formulated in various forms, such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injections of sterile injectable solutions according to the usual methods for each purpose of use. It may be used in combination, and may be administered orally, or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Such pharmaceutical compositions may further include a carrier, excipient, or diluent, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil And the like.

In addition, the pharmaceutical composition of the present invention may further contain a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, and the like.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient of the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per body weight daily or It can be administered every other day or divided into 1-3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount is not limited by any method.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to target cells.

In the present invention, the "subject" is not particularly limited, but includes, for example, a human, a monkey, a cow, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, a mouse, a mouse, a rabbit, or a guinea pig And, preferably, a mammal, more preferably a human.

In addition, the present invention provides a functional food for preventing or improving cancer, including the compound of Formula (I) or a pharmaceutically acceptable salt thereof, and TRAIL.

The TRAIL is preferably a recombinant human TRAIL, for example, as TRAIL of human origin.

The cancer is stomach cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer , Cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer and bone marrow cancer, preferably selected from the group consisting of, and more preferably acute T lymphocytic leukemia. Do.

The health functional food of the present invention can be used in various ways such as foods and beverages effective in preventing and improving cancer, preferably acute T lymphocytic leukemia.

Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules or beverages. .

In general, the active ingredient of the present invention may be added in an amount of 0.01 to 15% by weight of the total food weight, and the health beverage composition may be added in an amount of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional food of the present invention may contain the compound as an essential component in the indicated ratio as an additional component, as well as food supplementary additives acceptable for food, such as natural carbohydrates and various flavoring agents.

Examples of the natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and conventional sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

As the flavoring agent, taumatin, rebaudioside A, glycyrrhizin, saccharin, aspartame, and the like may be used. The proportion of the flavoring agent is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention.

In addition to the above, the health functional food of the present invention includes a variety of nutrients, vitamins, minerals, synthetic flavors, and flavoring agents such as natural flavors, colorants and thickeners, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juices, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is generally selected from 0.01 to about 20 parts by weight per 100 parts by weight of the active fraction of the present invention.

In addition, the present invention provides a method for promoting induction of apoptosis in a cell comprising treating the cell with a compound of the following formula (I) or a pharmaceutically acceptable salt thereof, and TRAIL in vitro.

In the induction promoting method, processes such as cell culture and reagent treatment may be performed as known to those of ordinary skill in the art, and the treatment concentration of the reagent is BS described in the specification of the present invention. The experimenter will be able to adjust the concentrations of -181 and rTRAIL.

The TRAIL is preferably a recombinant human TRAIL, for example, as TRAIL of human origin.

The cell may be a T cell, and includes, for example, a primary T cell isolated from a living body, a T cell line, a transformed T cell line, and the like.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples describe one preferred embodiment of the present invention, and the scope of the present invention is not limited and interpreted by the matters described in the following examples.

[Example]

1. Materials and methods

1.1. Reagents and antibodies

BS-181 was purchased from Selleckchem (Houston, TX, USA). Recombinant human TNF-related apoptosis-inducing ligand (rTRAIL) was purchased from PeproTech (London, UK). The human TRAIL ELISA kit was purchased from Koma Biotech (Seoul, Korea), 3,3'-dihexyloxacarbocyanine iodide (DiOC ₆ ), 4',6-diamidino-2-phenylindole (DAPI) and 3-(4,5- dimethythaizol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and paclitaxel (PTX), a pre-stop agent, were purchased from Sigma (St. Louis, MO, USA). The late G ₁ -stopper, hydroxyurea (HU), was purchased from Calbiochem (san Diego, CA, USA), the ECL western blot kit was from Amersham (Arlington Heights, IL, USA), and the Immobilon-P membrane was from Millipore Corporation. (Bedford, MA, USA). Anti-caspase-3 antibodies are from BD Transduction Laboratories (Franklin, NJ, USA), anti-poly (ADP-ribose) polymerase (PARP), anti-BCL-2, anti-BCL-XL, anti-CDK1, anti- CDK2, anti-CDK7, anti-Cyclin H, and anti-FADD antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In addition, anti-BID, anti-caspase-8, anti-caspase-9, anti-p-CDK1 (Tyr-15), anti-p-CDK1 (Thr-161), anti-p-CDK2 (Thr-160) , Anti-death receptor 5 (DR5), anti-p-retinoblastoma (Rb) (Ser-795), anti-p-Rb (Thr-821/826), and anti-TRAIL antibodies are Cell Signaling Technology (Beverly, MA, USA). The anti-BAK (Ab-1) antibody was purchased from Calbiochem (San Diego, CA, USA), and the anti-BAG3 antibody was purchased from Abcam (Cambridge, MA, USA).

1.2. Cell culture

Human acute leukemia Jurkat T cell clone wild-type A3, Fas-associated death domain (FADD)-deficient Jurkat T cell clone I2.1, caspase-8-deficient Jurkat T cell clone I9.2, U937 and human cervical cancer HeLa cells Was purchased from ATCC (Manassas, VA, USA) and used. In the culture of cells, 10% FBS, 20 mM HEPES (pH 7.0), 50 μM 2-mercaptoethanol and 100 μg/ml gentamicin were added to RPMI 1640 medium (Hyclone, Gaithersburg, MD, USA). HeLa cells were used by adding 10% FBS, 20 mM HEPES (pH 7.0) and 100 μg/ml gentamycin to DMEM (Hyclone). Jurkat T cell clones JT/Neo and JT/BCL-2 cells stably transferred to the empty vector (JT/Neo) or BCL-2 expression vector (JT/BCL-2) were obtained by Dr. Dennis Taub (Gerontology Research Center, NIA/NIH, Baltimore, MD, USA). In the culture of these JT/Neo and JT/BCL-2 cells, RPMI 1640 medium containing 10% FBS, 20 mM HEPES (pH 7.0), 50 μM β-mercaptoethanol, 100 μg/ml gentamicin and 400 μg/ml G418 was used. Used [44].

1.3. Cytotoxicity measurement

The cytotoxicity of BS-181 was measured by the MTT method [45].

1.4. Flow cytometry

Cell cycle analysis of Jurkat T cells treated with BS-181 was performed using a flow cytometer (FACS Calibur, BD Sciences, San Jose, CA, USA) according to the method [46] used in previous studies. In addition, analysis of necrotic cells using Annexin V-FITC apoptosis kit was performed according to the previous study method [46]. In addition, cells treated with BS-181 were first stained with FITC-Annexin V/DAPI using DAPI solution (1 μM) instead of PI solution of Annexin V-FITC apoptosis kit, and then fixed with 1% paraformaldehyde. , Subsequently, RNA was removed by treatment with 50 μg/ml RNase, and the permeability of the cell membrane was increased with 0.025% Digitonin, followed by secondary staining with PI (25 μg/ml). The stained cells were analyzed with a flow cytometer (Attune NxT, ThermoFisher Scientific, Waltham, MA USA) to analyze the cell cycle distribution pattern according to PI staining of early apoptotic cells stained only with FITC-Annexin V and not stained with DAPI. I did.

Loss of mitochondrial membrane potential (Δψm) accompanying BS-181 treatment was performed according to DiOC ₆ staining [47,48]. BAK activation according to BS-181 treatment was performed using the method [49] of the previous study. In addition, for the identification of TRAIL molecules expressed on the surface of the cells treated with BS-181, cells (5 × 10 ⁶ ) were recovered, washed with 1 × PBS + 2% FBS solution, and then anti-TRAIL antibody ( 1: 250) at 4° C. for 45 minutes, followed by Alexafluor 488-labeled anti-rabbit IgG (1: 500) at 4° C. for 30 minutes, fixed with 1% paraformaldehyde/PBS solution, and then cells were After washing, TRIL-positive cells were analyzed by FACS Calibur (BD Science, Franklin Lakes, NJ, USA).

1.5. Cell Lysate Preparation and Western Blot Analysis

Cell lysates were prepared by lysing 5 × 10 ⁶ Jurkat T cells in 300 μl of lysis buffer as described in previous studies [46]. The obtained cell lysate was taken in a certain amount in the range of about 20-25 μg in protein amount, electrophoresed using 4-12% NuPAGE gradient gel, and electrophoresed to the Immobilon-P membrane. Then, proteins on the membrane were analyzed using the ECL western blot kit. If necessary, the quantification of the protein bands on the Western blot was performed using ImageQuant TL software (Amersham, Arlington Heights, IL), and the quantification value of each band was standardized based on the quantification value of the GAPDH protein band.

1.6. Measurement of Soluble TRAIL by ELISA

Soluble TRAIL contained in the culture supernatant of Jurkat T cells treated with BS-181 was analyzed according to the manufacturer's experimental guidelines using an ELISA kit (Koma Biotech Inc. Seoul, Korea). All samples were performed in triplicate, and the optical density was measured using a microplate optical density meter, and the TRAIL concentration was measured by comparing it with a standard curve. The measurement results were expressed in pg/mL.

1.7. Statistical analysis

Unless otherwise specified, each result of this example presents an average of at least three separate experimental results. Experimental values were expressed as the mean value ± standard deviation of these experimental results. Statistical significance was calculated using Student's t-test. P values <0.05 were considered significant.

2. Results

2.1. Cytotoxic and apoptosis-inducing effects of BS-181 on Jurkat clones JT/Neo and JT/BCL-2 cells

To confirm that the cytotoxicity of BS-181 is due to the mitochondrial-dependent apoptosis pathway controlled by BCL-2, the cytotoxic effect of BS-181 on JT/Neo and JT/BCL-2 cells was compared. . Cytotoxicity in JT/Neo cells treated with BS-181 (5 ~ 20 μM) for 20 hours increased concentration-dependently, resulting in an IC ₅₀ value of 14.4 μM, whereas BCL-2 overexpressed JT/BCL- In 2 cells, the cytotoxicity of BS-181 was not observed (Fig. 1A). Likewise, when JT/Neo cells were treated with BS-181 at 10 and 15 μM for 24 hours, the proportion of sub-G ₁ cells increased to 23.2% and 54.8%, respectively, but in the case of JT/BCL-2 cells No increase in sub-G ₁ cells was observed (Fig. 1B). Under these conditions, G ₁ cell cycle arrest following BS-181 treatment was observed only in JT/BCL-2 cells, unlike JT/Neo cells. These results, in due to the BS-181 process is popto system G ₁ - so preferentially induced from the suspended cells, when in the G ₁ cell popto cis be blocked by BCL-2 overexpressing only by the BS-181 G ₁ -Suggests that an increase in quiescent cells can be observed.

In addition, JT/Neo cells treated with 15 μM BS-181 were stained with FITC-Annexin V and PI and analyzed by flow cytometry. As a result, initial apoptotic cells stained only with FITC-Annexin V and FITC-Annexin V and PI. The number of late apoptotic cells stained by all increased to 38.0% and 9.1%, respectively, whereas necrotic cells stained only with PI were hardly identified (FIG. 1C). Morphological changes appearing in apoptosis-progressing cells include cell contraction and exposure of phosphatidylserine to the outer layer of the cell membrane, whereas morphological changes in necrosis-progressing cells include cytoplasmic expansion, organelle expansion, and thus Concomitant plasma membrane destruction and the like are known [20]. When comparing the forward scatter distribution of early apoptotic cells stained only with FITC-Annexin V and late apoptotic cells stained with both FITC-Annexin V and PI in JT/Neo cells treated with BS-181, BS- In the early and late apoptotic cells induced by 181, a decrease in forward scatter was observed, reflecting a decrease in cell size in common (Fig. 1D). In contrast, in JT/BCL-2 cells, no increase in early or late apoptotic cells was observed even after BS-181 treatment.

These findings suggest that the cytotoxicity of BS-181 against Jurkat T cells is mainly due to apoptosis, which can be preferentially induced in G ₁ -stop cells, and the induction of apoptosis by BS-181 treatment is overexpressed. BCL-2, but G ₁ -arrest is not inhibited by overexpressed BCL-2.

2.2. ΔΨm reduction, BAK activation, caspase activation, soluble and membrane-bound TRAIL and DR5 expression induced by BS-181 treatment in JT/Neo and JT/BCL-2 cells

To investigate the relationship between apoptosis induced by BS-181 treatment and mitochondrial damage blocked by BCL-2 overexpression, the mitochondrial membrane potential of BS-181-treated JT/Neo and JT/BCL-2 cells ( Δψm) loss was analyzed by flow cytometry after DiOC ₆ staining. In the case of JT/Neo cells treated with 10 or 15 μM BS-181, the loss of Δψm was 27.0% and 66.0%, respectively, whereas in the case of JT/BCL-2 cells, the loss of Δψm by BS-181 was almost Not observed (Fig. 2A). With regard to the induction of Δψm, the polymerization of multidomain pro-epoptotic BCL-2 family proteins (BAK, BAX) that induces permeabilization of the outer mitochondrial membrane is important. However, anti-apoptotic BCL-2 family proteins such as BCL-2, BCL-XL and MCL-1 directly or directly with BH3-only pro-epoptotic BCL-2 family proteins (BAD, BID, BIM) Blocks BAK- or BAX-mediated damage to the outer mitochondrial membrane by indirect inactivation [21,22]. In JT/Neo cells treated with BS-181 at a concentration of 10 or 15 μM, the BAK activation ratio was 25.1% and 58.1%, respectively, but BAK activation was not observed in JT/BCL-2 cells (Fig. 2B ). Under these conditions, the BCL-2, BCL-XL and MCL-1 expression levels of JT/Neo cells treated with BS-181 were relatively constant (FIG. 2C). On the other hand, no activation of BIM [23] or increased expression of BAK was observed. In the induction of endogenous apoptosis, BAK activation and consequent loss of Δψm precedes first, resulting in the release of mitochondrial cytochrome c protein into the cytoplasm, as a result of which caspase-9/caspase-3 activation occurs. Consistent with the results [24-26], BAK activation, loss of Δψm, caspase-9/caspase-3 activation and cleavage of PARP were observed in JT/Neo cells treated with BS-181, whereas BS-181 induction No apoptosis reactions were observed in JT/BCL-2 cells. These findings demonstrate that the endogenous BAK-dependent mitochondrial-mediated apoptosis pathway blocked by BCL-2 is involved in the apoptosis of Jurkat T cells by BS-181 treatment.

The exogenous apoptosis pathway triggered by increased expression of DR5 or TRAIL inhibits the action of a truncated-BID (tBID, 15 kDa) resulting from caspase-8 activation and cleavage of BID (22 kDa) by active capase-8. Since BAK activation can be induced through [27,28], the expression levels of constituents of the exogenous apoptosis pathway were compared and investigated in JT/Neo and JT/BCL-2 cells treated with BS-181. As shown in FIG. 2D, in JT/Neo cells treated with BS-181, a significant decrease in the level of BID expression was observed, reflecting caspase-8 activation and cleavage to tBID, consistent with the increase in DR5 expression. In addition, in JT/Neo cells treated with BS-181, the levels of total cellular TRAIL, membrane-bound TRAIL, and secreted soluble TRAIL were found to increase, but in JT/BCL-2 cells, the total cellular TRAIL expression level was JT/Neo cells. Despite being about ~28 times higher than in the case of Neo cells, no such increase was observed after BS-181 treatment (Fig. 2D, E and F). In addition, in JT/BCL-2 cells, increased expression of DR5 was confirmed by BS-181 treatment, but caspase-8 activation, BID cleavage, caspase-9/caspase-3 activation, and PARP cleavage were all in overexpressed BCl-2. Blocked by and not observed.

These results suggest that the endogenous mitochondrial-dependent apoptosis response induced in JT/Neo cells by BS-181 treatment is subsequently linked to the exogenous apoptosis response activated by TRAIL/DR5-upregulation. .

2.3. Late G on caspase-8 activation, BID cleavage, BAK activation, loss of Δψm and caspase-9 activation induced by BS-181 in JT/Neo cells _One -Effect of hydroxyures (HU) or prometaphase-stopper paclitaxel (PTX)

To investigate whether the apoptosis phenomena induced by BS-181 treatment is associated with G ₁ -arrest accompanying the inhibition of CDK7 activity, CDK1 (Thr-161) and CDK2 (Thr-160) phosphorylated by CDK7 ) [29], and the effect of BS-181 on the phosphorylation of Rb [30,31], the phosphorylation substrate of CDKs, was compared in JT/Neo and JT/BCL-2 cells.

G ₁ -arrest by BS-181 treatment was confirmed only in JT/BCL-2 cells, but not only activated phosphorylation of CDK2 (Thr-160) and CDK1 (Thr-161) by the action of CDK7, but also the action of CDK1/2 It was found that phosphorylation of Rb (Ser-795) by the action of Rb (Thr-821/826) and CDK4/6 was inhibited in common in JT/BCL-2 cells and JT/Neo cells (Fig. 3). Under these conditions, the levels of G ₁ -cyclins (cyclin D1, cyclin D3) and G ₁ -CDK (CDK4, CDK2), and M-CDK and M-cyclin CDK1 and cyclin B1 were also decreased. JT/ Compared to Neo cells, JT/BCL-2 cells showed a greater tendency to decrease. These findings indicate that G ₁ -arrest following BS-181 treatment observed in JT/BCL-2 cells was due to the failure of Rb hyperphosphorylation by CDKs in the presence of BS-181 acting as a CDK7 inhibitor.

In order to further investigate whether the induction of apoptosis in JT/Neo cells by BS-181 treatment is due to G ₁ -cell cycle arrest, the enzyme activity of ribonucleotide reductase, which converts deoxyribonucleotides required for DNA synthesis, from ribonucleotides, is inhibited. Through complex treatment with hydroxyurea (HU) [32,33], which is known to stop cell cycle progression in late G ₁ , or by complex treatment with paclitaxel (PTX) [39], a prometaphase-stopper, late G ₁ by HU It was investigated whether -stopping could promote BS-181-mediated induction of apoptosis, and whether prometaphase-stopping by PTX could inhibit BS-181-mediated apoptosis. As a result, compared to the case of treatment with 15 μM BS-181 alone, the sub-G ₁ ratio increased by 2.0 times and the loss of Δψm increased by 2.0 times in the case of complex treatment with 15 μM BS-181 + 1 mM HU (Fig. 4A And B). On the other hand, it was found that the sub-G ₁ ratio and the loss of Δψm were rather reduced in the case of complex treatment with 15 μM BS-181 + 0.5 μM PTX (FIG. 4C and D).

In addition, JT/Neo cells treated with 15 μM BS-181 were first stained with FITC-Annexin V/DAPI and then fixed with paraformaldehyde, followed by treatment with RNase, followed by secondary staining with propidium iodide (PI). At this time, as a result of investigating the cell cycle according to PI staining for early apoptotic cells stained only with FITC-Annexin V but not stained with DAPI, cells stained with FITC-Annexin V were apoptotic sub-G ₁ 40.1% of cells, 35.4% of G ₁ cells, and 13.8% and 9.6% of S and G ₂ /M cells, respectively (E of Fig. 4).

These results indicate that the apoptotic response induced by BS-181 treatment in JT/Neo cells was arrested in late G ₁ phase due to cell cycle progression failing to enter S phase due to inhibition of CDK-mediated Rb phosphorylation. It confirms that it is first derived from.

2.4. Apoptosis by BS-181 in Jurkat clone wild-type A3, FADD-deficient Jurkat clone I2.1 and caspase-8-deficient Jurkat clone I9.2.

To further investigate the correlation between endogenous mitochondrial-dependent apoptosis induced by BS-181 and exogenous TRAIL/DR5-dependent apoptosis, cells of BS-181 were targeted for A3, I2.1 and I9.2 cells. The toxicity and the ability to induce apoptosis were compared. When A3 cells were treated with BS-181 at 5, 10, and 15 μM for 20 hours, the cell viability decreased to 92%, 67% and 46%, respectively, whereas I2.1 and I9.2 cells under the same treatment conditions. The viability of BS-181 was not significantly affected by the cytotoxicity of BS-181 up to the concentration of 10 μM, and the cell viability was slightly reduced at the concentration of 15 μM, but decreased to a much lower degree than that of A3 cells (Fig. 5A ).

In A3, I2.1 and I9.2 cells, the proportion of sub-G ₁ cells induced by 15 μM BS-181 treatment was 47.2%, 13.4% and 13.2%, respectively, and Δψm loss was 46.0%, respectively. , 14.4% and 16.0% (Fig. 5B and C). Under the same conditions, the BAK activation rates of A3, I2.1, and I9.2 cells were 44.3%, 15.8% and 16.3%, respectively (Fig. 5D). Since FADD-deficient I2.1 cells and caspas-8-deficient I9.2 cells are known to be cells that do not cause exogenous apoptosis, these findings suggest that BS-181 treatment in I2.1 and I9.2 cells. It is suggested that endogenous BAK-dependent apoptosis induced by BS-181 can also occur independently, independent of exogenous apoptosis induced by the treatment of BS-181.

In addition, to investigate the cell surface expression of TRAIL in A3, I2.1, and I9.2 cells treated with 15 μM BS-181, staining by immunofluorescence and analysis by flow cytometry showed that TRAIL was found on the surface of A3 cells. The expression level of was remarkably increased, but the expression of TRAIL on the cell surface of I2.1 and I9.2 did not increase very much (E of FIG. 5). On the other hand, as a result of investigating the apoptosis pathway induced by treatment with 15 μM BS-181 by Western blotting, in the case of A3 cells, caspase-8 activation and BID cleavage were caused by 15 μM BS-181 treatment, but I2 In the case of .1 and I9.2 cells, this apoptotic reaction did not occur (FIG. 5F). In addition, caspase-9/caspase-3 activation and PARP cleavage reactions were also observed at significantly lower levels in I2.1 and I9.2 cells than in A3 cells. The increase in the expression level of DR5 by BS-181 treatment was also more pronounced in A3 cells than in I2.1 and I9.2 cells. These results show that the expression levels of TRAIL and DR5 are signals of the exogenous apoptosis pathway. It is also consistent with previous studies that reported positive regulation by delivery [35].

As a result, current studies show that in Jurkat T cells, treatment with BS-181 (15 μM) may induce BAK-dependent mitochondrial apoptosis apart from the induction of exogenous apoptosis, but that of BS-181. It has been shown that cytotoxicity is mostly caused by exogenous apoptosis by TRAIL/DR5-upregulation.

To further investigate the importance of exogenous apoptosis related to the cytotoxicity of BS-181 on Jurkat T cells, A3, I2.1 and I9. The effect of addition of exogenous rTRAIL (1.0-4.0 ng/ml) on BS-181 cytotoxicity in 2 cells was investigated. As a result of MTT analysis, the synergistic effect of rTRAIL on the cytotoxicity of BS-181 appeared only in A3 cells, and was not observed in I2.1 and I9.2 cells (FIG. 6A, B and C). This shows that exogenous TRAIL/DR-mediated apoptosis, which can be augmented by rTRAIL, is a very important mechanism involved in the cytotoxicity of BS-181 in Jurkat T cells.

In order to investigate the synergistic mechanism of the combined treatment of BS-181 and rTRAIL, the effect of rTRAIL on exogenous apoptosis induced by BS-181 treatment in A3 cells was investigated. As shown in Fig. 7A, when A3 cells were treated with 10 μM BS-181 and 10 μM BS-181 + 1.0 ng/ml rTRAIL for 15 hours, the cell viability decreased to 73% and 54%, respectively. It was found to be 91% when only 1.0 ng/ml rTRAIL was used. At this time, the proportions of sub-G ₁ cells were 12.1%, 40.5%, and 18.9%, respectively, Δψm loss was 10.7%, 46.7% and 24.2%, respectively, and the BAK activation rates were 11.7%, 38.4%, and 13.9%, respectively. (Fig. 7 B, C and D).

In addition, as shown in E of FIG. 7, caspase-8 activation and BID cleavage induced by BS-181 treatment were significantly increased by complex treatment with rTRAIL, similar to caspase-9/capase-3 activation. Moreover, upregulation of DR5 expression level, which acts as an upstream signal of the BAK-mediated mitochondrial damage pathway induced by BS-181 treatment, was further enhanced by complex treatment with rTRAIL.

These results show that the synergistic effect of rTRAIL on the cytotoxicity of BS-181 in Jurkat A3 cells promotes exogenous TRAIL/DR5-mediated apoptosis signaling and thus BAK-dependent mitochondrial damage-mediated induction of apoptosis. It proves that it is caused by

2.5. Comparison of synergistic effects of rTRAIL on BS-181 cytotoxicity in malignant tumor cells and normal human T cells

To investigate whether the cytotoxic effect of BS-181 shows differences between malignant tumor cells and normal cells, and to clarify whether the selective cytotoxicity of BS-181 against malignant tumor cells can be enhanced by complex treatment with rTRAIL. , Cytotoxicity of BS-181 against malignant tumor cells (Jurkat A3, U937 and HeLa) and normal human peripheral blood T cells (unstimulated or PHA-stimulated human peripheral T cells) in the absence and presence of rTRAIL, respectively. It was investigated by the MTT method.

The IC ₅₀ values of BS-181 for Jurkat A3, U937 and HeLa cells were 14.5, 16.4 and 17.3 μM, respectively, but the IC of BS-181 against unstimulated or PHA-stimulated human peripheral blood T cells. _{The 50} values were 43.9 and 18.9 μM, respectively (Table 1). When comparing the synergistic effect of rTRAIL (1-4 ng/ml) on these BS-181 cytotoxicity between malignant and normal T cells, the synergistic effect of rTRAIL on BS-181 cytotoxicity was found in Jurkat A3, U937 and HeLa. Remarkably, in the case of the complex treatment of rTRAIL at a concentration of 4 ng/ml, it was found that the IC ₅₀ values of BS-181 for Jurkat A3, U937 and HeLa were lowered to 3.2, 11.4 and 13.0 μM, respectively (A in FIG. 8 , B and C). Under these conditions, in the case of PHA-stimulated human peripheral blood T cells, there was no synergistic effect of rTRAIL (1-4 ng/ml) on the cytotoxicity of BS-181 (FIG. 8D).

[Table 1] Inhibitory effect of BS-181 on IL-2-dependent proliferation of normal human-derived peripheral blood T cells and PHA-stimulated peripheral blood T cells and proliferation of tumor cells

The above study results show that the cytotoxicity of BS-181, which is elevated by the combination with rTRAIL, is highest in Jurkat T cells, a T-ALL cell line. In conclusion, these results show that the anticancer activity of the CDK7 inhibitor BS-181 is mainly induced by exogenous TRAIL/DR5-dependent apoptosis, and the combination of BS-181 and rTRAIL is promising as a chemotherapy for T-ALL. Suggests that you can.

Claims (11)

Acute T lymphoblastic leukemia (T-cell acute lymphoblastic leukemia, T-) containing a compound of the following formula (I) or a pharmaceutically acceptable salt thereof, and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) as active ingredients ALL) for the prophylaxis or treatment of pharmaceutical composition:

(Ⅰ) The pharmaceutical composition according to claim 1, wherein the TRAIL is a recombinant human TRAIL. delete delete Compounds of the following formula (I), or a pharmaceutically acceptable salt thereof, and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) of T-cell acute lymphoblastic leukemia (T-ALL) Dietary supplements for prevention or improvement:

(Ⅰ) The health functional food according to claim 5, wherein the TRAIL is a recombinant human TRAIL. delete delete In vitro ( in vitro ), T cells (T-cell) containing a compound of the formula (I) or a pharmaceutically acceptable salt thereof, and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) treatment How to promote the induction of apoptosis of T-cells:

(Ⅰ) The method of claim 9, wherein the TRAIL is a recombinant human TRAIL. delete

Images