WO2010064422A1 - Method for killing tumor by photosensitization treatment under conditions where histone is highly acetylated - Google Patents

Abstract

Disclosed is a therapeutic agent for cancer, which comprises a histone deacetylase inhibitor as an active ingredient and is intended to be used in combination with a PUVA therapy.  Also disclosed is a therapeutic agent for cancer, which comprises a combination of a histone deacetylase inhibitor and a photosensitizing agent and is intended to be used in combination with a PUVA therapy.  Preferred examples of the histone deacetylase inhibitor include sodium butyrate, trichostatin A (TSA), suberoylanilide hydroxamic acid (Zolinza/Volinostat/SAHA), valproic acid, Belinostat/PXD101, ITF2357, CRA-24781/PCI24781, Romidepsin/FK228/Depsipeptide, Phenylbutyrate/VP-101/EL-532, Pivanex/AN-9, SB939, MS-275/SNDX-275/entinostat, MGCD-0103 and JHJ-26481585.

Description

Tumor killing by photosensitization in histone hyperacetylated state

Related Application This application claims priority based on Japanese Patent Application No. 2008-307283 (filed on Dec. 2, 2008), the contents of which are incorporated herein by reference.

TECHNICAL FIELD The present invention relates to cancer photochemotherapy.

Photosensitization therapy is photochemotherapy using a combination of photosensitizer and ultraviolet light. PUVA therapy, which is a typical example, is a photochemotherapy that uses psoralen and long-wavelength ultraviolet (UVA) in combination, and has so far been highly effective against skin diseases such as common vitiligo, psoriasis, and atopic dermatitis. , Have been used a lot.

A photosensitizer is a substance that can be excited by absorbing light and transfer its energy to another substance to advance a photochemical reaction. A typical example of a photosensitizer is psoralen. This is a component contained in citrus fruits and is a tricyclic aromatic compound having a phototherapeutic action. Since psoralen has a planar structure, it can be intercalated into DNA at sites where pyrimidine-purines are alternately present, particularly TA sites (Hearst JE, Isaacs ST, Kanne D, et al. Q Rev Biophys 1984; 17: 1-44). After UVA (320-400 nm) irradiation, the intercalated complex is activated and an adduct is formed on the pyrimidine base in the cellular DNA. If there is an available pyrimidine at the appropriate position on the opposite side of the chain, the second UVA photon then binds to the free psoralen side of the chain and undergoes inter-strand crosslink (ICL). (Cimino GD, Gamper HB, Isaacs ST, et al. Annu Rev Biochem 1985; 54: 1151-93).

Since PUVA-induced ICL in chromosomal DNA induces anti-proliferative effects useful for skin treatment, psoralen + UVA (PUVA) is used in several hyperproliferative skin diseases such as psoriasis and cutaneous T-cell lymphoma Used for treatment (Stern RS. N Engi J Med 2007; 357: 682-90). There is also a report that PUVA showed cell killing effect (apoptosis) in cultured cell lines (Yoo EK, Rook AH, Elenitsas R, et al. J Invest Dermatol 1996; 107: 235-42; Canton M, Caffieri S, Dall'Acqua F, et al. PEBS Lett 2002; 522: 168-72), and cell killing power is not so strong compared to anticancer drugs and radiation irradiation, so that PUVA therapy can be applied to cancer treatment so far Was not considered.

The references cited in this specification are as follows. All the contents described in these documents are incorporated herein by reference.

The object of the present invention is to provide a photosensitizing method for cancer by enhancing the efficacy of PUVA therapy.

The present inventors have found that a very strong tumor cell killing effect can be obtained by using a HDAC inhibitor in combination with PUVA therapy. That is, the present invention provides a cancer therapeutic agent for use in cancer photochemotherapy comprising a combination of a histone deacetylase inhibitor and a photosensitizer. Preferably the cancer photochemotherapy is PUVA therapy. Preferably, the histone deacetylase inhibitor is sodium butyrate, valproic acid, Phenylbutyrate / VP-101 / EL-532, Pivanex / AN-9, OSU-HDAC42, trichostatin A, suberoylanilide hydroxamic acid (Zolinza / Volinostat / SAHA), LAQ-824, Panobinostat / LBH-589, CRA-24781 / PCI24781, Belinostat / PXD101, Tubacin, Oxamflatin, ITF2357, SB939, R306465 / JNJ-16241NST-275MS -0103, Pimeric diphenylamide, Romidepsin / FK 28 / Depepteptide, Apicidin, Trapoxin A, HC-Toxin, NCH-51, Alpha-mercaptoketone 19y / KD5150, 5- (Trifluoroacetoyl), 4-Citropide, 2-Ciboxide, 2-Ciboxide Selected from electrophilic ketones. In another aspect, the present invention provides an inhibitor of interstrand cross-link repair comprising a histone deacetylase inhibitor as an active ingredient.

In another aspect, the present invention provides a method of treating cancer by photochemotherapy, wherein the method comprises administering to a patient a histone deacetylase inhibitor and a photosensitizer and then treating the site. Irradiation with UVA. Preferably, the photosensitizer is psoralen. In yet another aspect, the present invention provides a method of inhibiting repair of interstrand crosslinks caused by UVA irradiation in a patient undergoing cancer photochemotherapy, wherein the method comprises a histone deacetylase in the patient. Administration of an inhibitor. Preferably, the cancer photochemotherapy is PUVA therapy.

According to the present invention, cancer cells can be killed very efficiently and in a short time, which is promising as a new cancer photosensitization treatment method. In addition, in terms of performing light irradiation, only cancer cells can be targeted, and there is no worry of damaging normal cells, so it can be expected to prevent secondary cancer caused by treatment.

FIG. 1 shows cell viability when melanoma cells were treated with PDAC after being treated with an HDAC inhibitor. FIG. 2 shows the acetylation state of histone H3 when melanoma cells are treated with an HDAC inhibitor. FIG. 3 shows cell viability when A549 and MCF-7 cells were treated with HDAC inhibitors and then treated with PUVA. FIG. 4 shows cell viability when melanoma cells were treated with HDAC inhibitor and then treated with 5-MOP + UVA. FIG. 5 shows the induction of apoptosis when melanoma cells are treated with a combination of an HDAC inhibitor and PUVA. FIG. 6 shows cell viability when melanoma cells were treated with combinations of HDAC inhibitors and various anticancer agents. FIG. 7 shows the repair kinetics of CPD when melanoma cells are treated with HDAC inhibitors and then irradiated with UVC. FIG. 8 shows cell viability when melanoma cells were treated with an HDAC inhibitor and then irradiated with UVC. FIG. 9 shows the time course of cell viability and histone H3 acetylation level when melanoma cells were treated with a HDAC inhibitor and then cultured in a medium containing no HDAC inhibitor. FIG. 10 shows cell viability when cutaneous T-cell lymphoma was treated with a combination of an HDAC inhibitor and various anticancer agents.

In the cells treated with sodium butyrate (SB), which is an HDAC inhibitor, the present inventors have shown that the photosensitizing action (PUVA) of UVA and 8-methoxypsoralen (8-MOP) is high. It has been found that it induces cell death (apoptosis) effectively in human melanoma cells, skin squamous cell carcinoma cells, breast cancer cells, lung epithelial cancer cells, and skin T cell lymphomas that are induced by sensitivity and resistant to anticancer agents. , Clarified the mechanism.

DNA is wound around a histone octamer composed of four types of core histones to form a chromatin structure. The level of histone acetylation is regulated by histone acetylase (HAT) and histone deacetylase (HDAC). Histone acetylated by HAT unwinds an aggregated chromatin structure and allows various molecules to access DNA, thereby activating gene transcription.

Histone deacetylase (HDAC) inhibitors are attracting attention as promising new types of anti-tumor agents for treating solid tumors and hematological malignancies (Marks PA, Miller T, Richon VM. Curr Opin Pharmacol 2003) ; 3: 344-51; Marks PA, Richon VM, Miller T, et al. Adv Cancer Res 2004; 91: 137-68; Mai A, Massa S, Rotili D, et al. Med Res Rev 2005; 25: 261 -309). HDAC is an enzyme that controls the acetylation state of core nucleosome histones by removing the acetyl group from the amino terminal residue. When HDAC is inhibited, acetylated histone accumulates due to the action of histone acetyltransferase. This weakens histone-DNA interactions and consequently increases the accessibility of transcription factors to DNA, causing changes in the expression of several proteins involved in the control of cell cycle, differentiation and apoptosis (Marks PA, Richon VM, Rifkind RA. J Nad Cancer Inst 2000, 92: 1210-6). For example, pro-apoptotic factors (eg, Bak, Bax, and Bim) and anti-apoptotic factors (eg, Bcl-2, Bcl-XL, XIAP, and Mcl-1) are up-regulated and down-regulated by HDAC inhibitors, respectively. It is regulated and is considered to play an important role in antitumor activity (Zhang XD, Gillespie SK, Borrow JM, et al. Mol Cancer Ther 2004; 3: 425-35).

It is also drawing attention to treat cancer using HDAC inhibitors in combination with radiation and anticancer drugs (Karagiannis TC, El-Osta A. Cell Cycle 2006: 5: 288-95; Camphausen K, Tofilon PJ. J Clin Oncol 2007; 25: 4051-6). It has been reported that various HDAC inhibitors (sodium butyrate (SB), trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), valproic acid, etc.) enhance the cell killing effect of radiation and anticancer agents. ing. However, the molecular mechanism has not been fully elucidated.

The present inventors have reported that the most widely studied HDAC inhibitors sodium butyrate (SB) and trichostatin A (TSA) are skin malignant melanoma cell lines SK-MEL-28, G-361, squamous cell carcinoma of the skin It has been found that the effect of PUVA on killing apoptotic cells can be remarkably enhanced in cells HSC-1, breast cancer cell MCF-7, lung epithelial cancer cell A549, cutaneous T cell lymphoma HH, HUT-78. As shown in the examples below, treatment with PUVA or SB alone had little effect on the cell viability of SK-MEL-28, but the combination treatment of SB and PUVA was prominent within 24 hours. Apoptosis was induced. The degree of enhancement of cell killing effect induced by SB was much higher in combination with PUVA than in combination with anticancer agents. In addition, the degree of enhancement of the cell killing effect on cutaneous T cell lymphomas HH and HUT-78 was much higher in combination with PUVA than in combination with anticancer agents. These were the same when the HDAC inhibitor was TSA. The number of double-strand breaks formed in the process of repairing PUVA-induced interstrand crosslinks (ICL) in chromosomal DNA is significantly reduced in cells pretreated with SB, which indicates the ability to repair ICL It was suggested that was reduced by SB.

It is known that repair of psoralen interstrand crosslinks (ICL) generated by PUVA involves multiple repair pathways such as nucleotide excision repair (NER) and homologous recombination repair. In particular, nucleotide excision repair has been reported to be involved in the early stages of ICL repair, such as recognition of ICL damage and excision of damaged sites. In the present invention, bromodeoxyuridine incorporation and the formation of proliferating cell nuclear antigen (PCNA) repair focus associated with nucleotide excision repair (NER) in ICL removal after PUVA treatment is observed in cells pretreated with SB. Was not observed. This indicates that SB reduces DNA repair, especially nucleotide excision repair (NER), which contributes to the enhanced cell killing effect of PUVA. Also, the repair rate of UV-induced cyclobutane pyrimidine dimer (well known as photo-damage repaired by NER) was much slower in cells pretreated with SB than in untreated cells. This result indicates that SB promotes repair of DNA damage induced by ultraviolet irradiation (UVC) (Smerdon et al., J. Biol. Chem., 1982; 257 (22), 13441-13447). ; Shows the opposite result from Ramanathan and Smerdon, J. Biol. Chem., 1989; 264 (19), 10026-11034). As the above paper argues, if the repair of DNA damage induced by ultraviolet light (UVC) is promoted by SB action, cell viability after combined UV irradiation and SB should recover. , No such event was observed (Figure 8). Therefore, the phenomenon found in the present invention is a surprising discovery by the present inventors.

While not intending to be bound by any theory, the present inventors have currently shown that the mechanism of enhanced cell death obtained by the present invention is the ICL repair pathway, particularly by pretreatment with HDAC inhibitors. It is thought that apoptosis was effectively induced as a result of inhibition of nucleotide excision repair and failure to repair DNA damage (ICL).

As the HDAC inhibitor used as an active ingredient of the cancer therapeutic agent of the present invention, any substance that inhibits histone deacetylase may be used. For example, sodium butyrate, valproic acid, phenylbutyrate / VP -101 / EL-532, Pivanex / AN-9, OSU-HDAC42, trichostatin A, suberoylanilide hydroxamic acid (Zolinza / Volinostat / SAHA), LAQ-824, Panobinostat / LBH-589, CRA-24781 / PCI24781 Belinostat / PXD101, Tubacin, Oxamflatin, ITF2357, SB939, R306465 / JNJ-16241199, MS-275 / SNDX-275 / enti ostat, MGCD-0103, Pimelic diphenylamide, Romidepsin / FK228 / Depsipeptide, Apicidin, Trapoxin A, HC-Toxin, NCH-51, Alpha-mercaptoketone 19y / KD5150,5- (trifluoroacetyl) thiophene-2-carboxamides, 2-Trifluoroacetylthiophene oxadiazoles CI-994, Chlamydocin, JHJ-264815585, electrophilic ketone, and the like. Preferably, the histone deacetylase inhibitor is sodium butyrate, trichostatin A (TSA), suberoylanilide hydroxamic acid (Zolinza / Volinostat / SAHA), valproic acid, Belinostat / PXD101, ITF2357, CRA-24781 / PCI24781 Romidepsin / FK228 / Depepteptide, Phenylbutyrate / VP-101 / EL-532, Pivanex / AN-9, SB939, MS-275 / SNDX-275 / entinostat, MGCD-0103, JHJ-26481585. All of these materials are commercially available or their structures are known and can be easily produced by those skilled in the art.

Examples of the photosensitizer used as an active ingredient of the cancer therapeutic agent of the present invention include psoralen and its derivatives. Psoralen and its derivatives have the following general formula:

[In the formula, each R independently represents hydrogen or a C _1-6 alkyl group or alkoxy group]
Can be expressed as Particularly preferred are 8-methoxypsoralen (mesoxalene), 5-methoxypsoralen and trimethylpsoralen (trioxalene). These photosensitizers are commercially available or can be easily produced by those skilled in the art.

The cancer therapeutic agent of the present invention can be formulated by methods known to those skilled in the art. For example, an HDAC inhibitor or a photosensitizer is added to a pharmaceutically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavor. It can be formulated by mixing in a unit dosage form generally required for pharmaceutical practice, in appropriate combination with agents, excipients, vehicles, preservatives, binders and the like. The HDAC inhibitor and the photosensitizer may be provided as separate packages, or may be provided as a kit combining these.

The cancer therapeutic agent of the present invention may be administered systemically by oral or parenteral administration, or may be administered locally. When the cancer therapeutic agent of the present invention is used in PUVA therapy, UVA can be irradiated to the site to be treated after the HDAC inhibitor and the photosensitizer are administered by oral or parenteral administration. Alternatively, UVA may be irradiated after applying the HDAC inhibitor and the photosensitizer to the site to be treated or immersing the site to be treated in the aqueous solution of the HDAC inhibitor and the photosensitizer. Alternatively, before irradiation with UVA, the HDAC inhibitor may be administered systemically and the photosensitizer may be administered locally, or conversely, the photosensitizer may be administered systemically and the HDAC inhibitor may be administered locally. Alternatively, after HDAC inhibitor and photosensitizer are administered by oral or parenteral administration, blood is removed from the patient and irradiated with UVA, or HDAC inhibitor is administered by oral or parenteral administration, and blood is then removed from the patient. After removal and treatment with PUVA, the blood may be returned to the patient.

The contents of all patents and references explicitly cited herein are hereby incorporated by reference.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

Materials and Methods (1) Cells and Cell Culture Conditions Cell lines SK-MEL-28 and G-361 derived from malignant pigmented melanoma were purchased from The Health Science Research Resources Bank (Japan). Squamous cell carcinoma cell HSC-1 was purchased from RIKEN Cell Bank. Human skin T-cell lymphomas HUT78 and HH were purchased from Riken Cell Bank and American Type Culture Collection (ATCC), respectively. Cells were maintained in medium appropriate for each cell (MEM, DMEM, RPMI) supplemented with 10% fetal bovine serum (FBS) and 100 units / ml penicillin / streptomycin at 37 ° C. under 5% CO ₂ .

(2) Treatment of melanoma by a combination of SB and PUVA SK-MEL-28 cells were treated with 8-methoxypsoralen (8-MOP: 10 ⁻⁷ −10 ⁻⁵ M) in a medium containing SB (0.1-3 mM). Then, the cells were cultured for a predetermined time, and UVA (1-5 J / cm ² ) was irradiated immediately after the medium was replaced with SB-free MEM. The UV irradiation intensity at the sample level was about 1.5 mW / cm ² , and the UVA irradiation of 5 J / cm ² required about 1 hour.

(3) Viability assay Cell viability was assessed by metabolism of fluorescein diacetate (FDA) (Wako, Japan). Cells were suspended in PBS containing FDA (0.1 mg / ml) and incubated at 37 ° C. for 15 minutes. After addition of propidium iodide (PI) (10 μg / ml), the fluorescence intensity was measured using a flow cytometer (FCM) (Epics XL; Coulter). Cells were divided into three populations by double staining with FDA and PI: (i) producing viable cells (FDA +, PI−), (ii) dead cells (FDA−, PI +), and (iii) causing apoptosis. Cells (FDA-, PI-). Evaluation of apoptosis by double staining with FDA and PI is equivalent to evaluation using annexin V staining (a highly specific index of apoptosis).

(4) Apoptosis assay Apoptosis was determined by staining with the DNA-binding fluorophore Hoechst 33258 to detect morphological changes in chromatin and measuring the percentage of apoptotic nuclei (sub-G ₁ fraction) by PI. . Caspase-3 activity was measured using an enzyme fluorescent substrate (Ac-Asp-Glu-Val-Asp-MCA).

(5) Detection of DNA double-strand break (DSB) DSB was detected using a biased sinusoidal gel electrophoresis (BSFGE) system (ATTO, Japan). The cells were suspended and solidified in 1% low melting point agarose immediately after treatment. The agarose plug was treated with 0.5 mg / ml protease K and 1 mg / ml ribonuclease A and electrophoresed on a 0.8% agarose gel. The gel was stained with ethidium bromide and photographed.

(6) DNA repair synthesis (DRS) assay Cells were grown to confluence in 60 mm dishes or 35 mm glass dishes and cultured for an additional 24 hours in serum-depleted MEM. This was treated with SB (3 mM) in MEM containing 0.5% FBS for 24 hours. Next, PUVA treatment was performed, and the cells were incubated with bromodeoxyuridine (BrdU) (10 μM) for a predetermined time (0-30, 30-60, 60-90, 90-120 minutes after PUVA treatment).

For flow cytometry analysis, cells fixed with ethanol (70%) were treated with 2N HCl for 15 minutes at room temperature. After blocking with 1% bovine serum albumin (BSA), secondary antibody (Jackson ImmunoResearch) conjugated with fluorescein isothiocyanate (FITC) together with primary antibody against BrdU (Santa Cruz Biotechnology) (1: 200) ( 1: 200). The increase in FITC intensity based on BrdU incorporation during each repair period was analyzed using a flow cytometer.

For immunofluorescence microscopy, cells fixed with 4% paraformaldehyde (PFA) were immersed in PBS containing 0.5% Triton X-100 at 4 ° C. for 3 minutes and treated with 2N HCl. This DNA denaturation step was performed only in the case of the BrdU incorporation experiment, and was not performed in the detection of the repair focus of the proliferating cell nuclear antigen (PCNA). After blocking with 1% BSA, incubated with primary antibody against BrdU (1: 200) or PCNA (Santa Cruz Biotechnology) (1: 200) and then with a secondary antibody conjugated with FITC (1: 200) . Nuclei were stained with PI (20 μg / ml). Images were acquired with a laser scan confocal microscope (LSM510, Carl Zeiss, Germany).

(7) Measurement of repair kinetics of cyclobutane pyrimidine dimer (CPD) For in-situ detection of CPD, cells cultured in a 35 mm glass bottom dish were treated with SB (3 mM) for 24 hours, and a polycarbonate ISOPORE membrane filter (pore) (Size: 3 μm) UVC (0.02 J / cm ² ) was irradiated through (Millipore) (main emission wavelength 254 nm) (ATTO, Japan). After irradiation, photoinjury was repaired by incubating with SB-free MEM for a predetermined time (0-24 hours). Cells were fixed with 4% paraformaldehyde, treated with 0.5% Triton X-100 in PBS, and treated with 2N HCl. After blocking with 1% BSA, it was incubated with a primary antibody against CPD (1: 1500) (Medical & Biological Laboratories, Japan) and then with a secondary antibody conjugated to FITC. Nuclei were stained with PI (20 μg / ml). Images were acquired with a laser scanning confocal microscope.

For detection of CPD by enzyme immunoassay (ELISA), cells were treated with SB (3 mM) for 24 hours and irradiated with UVC (0.002 J / cm ² ). After incubation with SB-free MEM for 24 hours, genomic DNA was extracted and denatured by boiling for 15 minutes. DNA (l μg / well) was added to a polyvinyl chloride 96 well plate pre-coated with protamine sulfate (0.003%).

After blocking with 2% FBS in PBS, the samples were combined with a primary antibody against anti-CPD (1: 2000) and then with a biotin-F (ab ′) ₂ fragment of anti-mouse IgG (Zymed) (1: 2000), Incubated. Next, peroxidase reaction was performed using o-phenylenediamine (0.4 mg / ml) in the presence of 0.02% H ₂ O ₂ . The reaction was stopped by adding H ₂ SO ₄ (2M), and the absorbance at 492 nm was measured with a spectrophotometer.

(8) Statistics Values are expressed as mean ± SD (n = 3-4). Data were analyzed by one-way ANOVA, and comparisons between groups were performed by Dunnett's t test. * P <0.05, ** p <0.01, and *** p <0.001 were considered statistically significant.

Results (1) Sodium butyrate (SB) enhances the cell killing effect of PUVA in SK-MEL-28 cells.
After SK-MEL-28 cells were treated with SB (0.1-3 mM) for a predetermined time (4-24 hours), the cells were treated with PUVA (8-MOP: 10 ⁻⁷ −10 ⁻⁵ M, UVA: 1-5J). / Cm ² ) and cell viability was measured by FDA assay. FIG. 1A shows cell viability as measured by FDA assay 24 hours after PUVA treatment. SK-MEL-28 cells were highly resistant to PUVA treatment. SB-PUVA treatment induced dramatic cell death, the extent of which was dependent on SB dose, SB treatment time, 8-MOP dose and UVA dose. When the time course of cell viability after treatment with SB (3 mM) -PUVA (8-MOP: 10 ⁻⁵ M, UVA: 5 J / cm ² ) was examined, the viability decreased rapidly in 8 to 24 hours. (FIG. 1B). No synergistic effect was observed when cells were treated in the reverse order (first with PUVA and then with SB).

Next, cells pretreated with another histone deacetylase inhibitor TSA (0.1-10 μM) for 24 hours were treated with PUVA (8-MOP: 10 ⁻⁵ M, UVA: 5 J / cm ² ). , Cell viability was measured after 24 hours. TSA also enhanced the cell killing effect of PUVA as in SB (FIG. 1C). Similar effects were also observed in melanoma cells G-361 other than SK-MEL-28 cells and squamous cell carcinoma cells HSC-1. (FIG. 1D).

The acetylation state of histone H3 when treated with SB was examined. The cells were treated with SB (0.1-3 mM) for a predetermined time (4-24 hours), and 500 μl of ice-cold lysis buffer (10 mM Tris-HCl, 50 mM sodium bisulfite, 1% Triton X-100, 10 mM MgCl ₂ , 8 .6% sucrose, pH 6.5). After homogenization, the nuclei were collected by centrifugation at 2,000 g for 5 minutes and washed twice with lysis buffer and once with TE (10 mM Tris-HCl, 13 mM EDTA, pH 7.4). The pellet was suspended in 150 μl HCl (0.25N) and incubated on ice for at least 1 hour. After centrifugation at 12,000 g for 5 minutes, the supernatant was mixed with 1.2 ml of acetone. After incubation overnight at 4 ° C., the aggregated material was collected by centrifugation at 12,000 g for 5 minutes and air-dried. This acid-soluble histone fraction was dissolved in 30 μl of H ₂ O and subjected to SDS-PAGE using about 20 μg of protein. The acetylation status of histone H3 was confirmed by Western blot using anti-acetyl-histone H3 (UPSTATE).

The results are shown in FIG. SB treatment increased the level of acetylated histone H3, the extent of which was dependent on (A) SB dose and (B) treatment time. In addition, even when it processed with TSA instead of SB, the pattern of the acetylation state of histone H3 was substantially the same.

Human breast adenocarcinoma cells (MCF-7) and human lung adenocarcinoma epithelial cells (A549) were treated with SB-PUVA in the same manner as in FIG. 1A. Briefly, cells were treated with SB (3 mM) for 24 hours, followed by PUVA treatment (8-MOP: 10 ⁻⁷ −10 ⁻⁵ M, UVA: 5 J / cm ² ). Cell viability 24 hours after treatment was assessed by FDA assay. Cell death after only PUVA treatment was slight, but pretreatment with SB significantly enhanced the cell killing effect of PUVA, consistent with the results obtained for SK-MEL-28 cells (FIG. 1A). (FIG. 3). That is, SB-PUVA treatment was able to effectively kill human breast adenocarcinoma cell MCP-7 and human lung adenocarcinoma epithelial cell A549.

SK-MEL-28 cells were treated with SB (3 mM) for 24 hours, and 5-methoxypsoralen (5-MOP) (Sigma-Aldrieh) (10 ⁻⁷ −10 ⁻⁵ M), a positional isomer of 8-MOP. Was used and treated with UVA (5 J / cm ² ) in the same manner as described above. Cell viability 24 hours after treatment was assessed by FDA assay. The cell killing effect of 5-MOP + UVA was significantly enhanced by pretreatment with SB (FIG. 4). That is, pretreatment with SB significantly increased the cytotoxicity induced by 5-methoxypsoralen + UVA. These results indicate that pretreatment with a histone deacetylase inhibitor effectively induces cell death by PUVA.

(2) The combination treatment of SB and PUVA induces apoptosis in SK-MEL-28 cells The induction of apoptosis in SK-MEL-28 cells by the combination treatment of SB and PUVA was examined. Cells pretreated with SB (3 mM) for 24 hours were treated with PUVA (8-MOP: 10 ⁻⁵ M, UVA: 5 J / cm ² ), and further incubated for a predetermined time (4-24 hours). Induction of apoptosis at each time, flow cytometry analysis by double staining with FDA and PI (FIG. 5A), detection of sub-G ₁ fraction (FIG. 5B), morphological evaluation of apoptotic cells using Hoechst 33258 ( FIG. 5C) was evaluated based on the measurement of caspase-3 activity using a fluorescent substrate (FIG. 5D) (** p <0.01, *** p <0.001).

As shown in FIG. 5, after treatment with SB-PUVA, the FDA (+) PI (−) compartment cell population undergoes FDA (−) PI (−) via FDA (−) PI (−) in a time-dependent manner. Moved to +). A time-dependent increase in the sub-G ₁ fraction was also observed. The number of chromatin-condensed cells increased in a time-dependent manner after SB-PUVA treatment, but did not increase after treatment with UVA alone or SB-UVA treatment. Caspase-3 activation was observed between 8 and 16 hours after treatment. Furthermore, z-vad-fmk, a total caspase inhibitor, clearly reduced cell death induced by SB-PUVA treatment. These results indicate that SB-PUVA treatment effectively induced typical caspase-dependent apoptosis in SK-MEL-28 cells.

(3) The enhancement of cell killing effect by the combination treatment of SB and PUVA is more dramatic than the combination of SB and anticancer agent.
Cells pre-cultured with SB (3 mM) for 24 hours were treated with various anticancer agents (bleomycin, etoposide, adriamycin, 5-fluorouracil, paclitaxel, and vincristine) for 24 hours. The dose ranges of anticancer drugs used here are based on literature descriptions (Kim MS, Blake M, Baek JH, et al. Cancer Res 2003; 63: 7291-300; Dowdy SC, Jiang S, Zhou XC, et al. Mol. Cancer Ther 2006,5: 2767-76; Sonnemann J, Kumar KS, Heesch S, et al. Int J Oncol 2006; 28: 755-66; Dong YB, Yang HL, Elliott MJ, et al. Cancer Res 2002; 62 : 1776-83). Cell viability was measured by FDA assay.

The results are shown in FIG. In any case, the combined treatment of SB and the anticancer agent resulted in an increase in cytotoxicity compared to the treatment with the anticancer agent alone, but the cell killing effect of the treatment with SB and PUVA was much greater than the combined treatment of SB and the anticancer agent. it was high. These results indicate that the enhancement of cell killing effect by the combination of SB and PUVA cannot be explained only by promoting the induction of apoptosis.

(4) SB reduces the number of DNA double strand breaks (DSB) formed by the PUVA treatment.
DNA damage induced by PUVA treatment is mainly due to psoralen interstrand crosslinks (ICL), which appears to be the main cause of cell killing effect induced by PUVA. That is, it is considered that SB reduced the repair efficiency of psoralen ICL, which led to significant cell death. DNA double-strand breaks (DSB) have been reported to occur as intermediates in psoralen ICL cell repair (Dronkert ML, Kanaar R. Mutat Res 2001; 486: 217-47). The extent of DSB formation after treatment with PUVA or SB-PUVA was examined using BSFGE as an indicator of the efficiency of ICL repair.

Cells pretreated with SB (0.5-3 mM) for 2-24 hours were treated with PUVA (8-MOP: 10 ⁻⁵ M, UVA: 5 J / cm ² ). Immediately after the PUVA treatment, DSB was obtained by BSFGE. When detected, clear DSB was observed. Pretreatment with SB reduced the number of DSBs produced, the extent of which was dependent on the dose and time of SB pretreatment. Also, when cells pretreated with TSA (0.1-30 μM) for 24 hours were treated with PUVA (8-MOP: 10 ⁻⁵ M, UVA: 5 J / cm ² ), the DSB after treatment with PUVA Production decreased in a dose-dependent manner. On the other hand, cells pretreated with SB (3 mM) for 24 hours were further cultured in SB-free medium for 0-24 hours, and then subjected to PUVA treatment (8-MOP: 10 ⁻⁵ M, UVA: 5 J / cm ² ). went. As a result, according to the length of time cultured in the SB-free medium, the formation of DSB decreased by SB was restored to the original level induced by PUVA. These results indicate that SB reduces ICL repair induced by PUVA.

(5) SB reduces DNA repair synthesis (DRS) after PUVA treatment.
It has been proposed that the removal of psoralen ICL involves the nucleotide excision repair (NER) pathway (Dronkert ML, Kanaar R. supra). The ability of NER in SB-treated cells after PUVA treatment was measured by DRS assay. Cells treated with SB (3 mM) for 24 hours in MEM containing 0.5% FBS were treated with PUVA (8-MOP: 10-5 M, UVA: 5 J / cm ² or 8-MOP: 10 ⁻⁴ M, UVA: 1 J / cm ² ). They were then incubated with BrdU (10 μM) for the indicated times (0-30, 30-60, 60-90, and 90-120 minutes after PUVA treatment). Using flow cytometry, the fluorescence intensity due to incorporated BrdU was measured.

As a result, the intensity of fluorescence from incorporated BrdU was significantly higher in PUVA-treated cells than in untreated cells for all times examined (0-2h). The degree of increase in fluorescence intensity did not differ greatly depending on the BrdU labeling time. On the other hand, when cells were pretreated with SB, no increase in fluorescence was observed after PUVA treatment. Furthermore, the repair focus of PCNA was examined with an immunofluorescence microscope. Cells pretreated with SB (3 mM) in MEM containing 0.5% FBS for 24 hours were treated with PUVA (8-MOP: 10 ⁻⁵ M, UVA: 5 J / cm ² ). After culturing for 1 hour, the cells were fixed. In the nucleus, it was possible to detect a clear focus of BrdU due to DRS after the PUVA treatment. However, such a focus could hardly be detected when cells pretreated with SB were treated with PUVA. These results show that the ability of SB to remove psoralen ICL is reduced by SB.

(6) SB reduces the repair rate of cyclobutane pyrimidine dimer (CPD) formed by UVC irradiation.
To further confirm that SB reduced NER, the kinetics of repair of CPD formed by local UVC exposure was examined. Cells treated with SB (3 mM) for 24 hours were partially irradiated with UVC (0.02 J / cm ² ) through a polycarbonate ISOPORE membrane filter. After incubation for the indicated time (0-24 hours), the cells were fixed. As a result, a clear CPD focus having a high fluorescence intensity was detected immediately after irradiation (0 h) in both SB pretreated cells and untreated cells (FIG. 7A). Without pretreatment with SB, the fluorescence weakened and the outline of the CPD focus became ambiguous after 24 hours of treatment. On the other hand, clear CPD focus was still detected in SB pretreated cells.

Cells with CPD focus with both clear contour and high fluorescence intensity were considered CPD positive (FIG. 7B, * p <0.05, ** p <0.01, *** p <0 .001). CPD-positive cells decreased more rapidly in SB-untreated cells than in SB-pretreated cells with repair time. Twenty-four hours after UV irradiation, approximately 15% of untreated cells and 60% of SB pretreated cells were CPD positive. Next, the cells treated with SB (3 mM) for 24 hours were irradiated with UVC (0.002 J / cm ² , no membrane filter). After culturing for 24 hours, the amount of CPD in the genomic DNA was examined by ELISA. CPD levels were significantly higher for SB pretreated cells than untreated cells (FIG. 7C). That is, in the SB pretreated cells, the repair of CPD induced by UVC irradiation (well known as light injury repaired by NER) was slower. These results further support the hypothesis that SB reduces the ability of NER.

Next, the effect of the combination of SB pretreatment and UVC was examined. SK-MEL-28 cells were treated with SB (3 mM) for 24 hours. Next, UVC (0.005-0.025 J / cm ² , no membrane filter) was irradiated. Cell viability 24 hours after treatment was assessed by FDA uptake assay. The results are shown in FIG. UVC irradiation reduced cell viability in a dose dependent manner. That is, pretreatment with SB significantly enhanced cell death induced by UVC. Furthermore, the UVC-induced cell death and the PUVA-induced cell death enhanced by SB pretreatment were more pronounced in the syngeneic strain lacking XPA in the cell line expressing XPA (an essential protein for NER). These results further support our hypothesis that SB reduces DNA repair, particularly the NER pathway.

As described above, DSB production during ICL repair is reduced by SB, but it recovers over time when incubated with SB after SB treatment. This suggests that the ability to NER has been restored. Then, next, it was investigated whether the effect of SB processing was reversible. SK-MEL-28 cells were treated with SB (3 mM) for 24 hours and further cultured in SB-free medium for a predetermined time. Next, PUVA treatment (8-MOP: 10 ⁻⁵ M, UVA: 5 J / cm ² ) was performed, and cell viability 24 hours after the treatment was evaluated by FDA assay. When cells were treated with PUVA 24 hours after SB treatment, dramatic cell death was induced. However, when cultured in SB-free medium prior to PUVA treatment, the cells gradually became resistant to PUVA (FIG. 9). This indicates that the decrease in NER ability by SB is reversible.

Next, SK-MEL-28 cells were treated with SB (3 mM) for 24 hours, and further cultured in SB free medium for a predetermined time. When the acetylation state of histone H3 was examined by Western blot analysis, highly acetylated histone H3 immediately after treatment with SB for 24 hours decreased with time, and the acetylation level of histone H3 was determined by viability. Well correlated.

FIG. 10 shows cell viability when skin T cell lymphoma cells HH and HUT78 were treated with HDAC inhibitors and then treated with PUVA or anticancer agents (FIG. 10). Even in these cells, the combination of PUVA showed a higher anticancer effect than the combination of HDAC inhibitor and anticancer agent treatment.

Claims (9)

1. A cancer therapeutic agent for use in cancer photochemotherapy comprising a combination of a histone deacetylase inhibitor and a photosensitizer.
2. The cancer therapeutic agent according to claim 1, wherein the cancer photochemotherapy is a PUVA therapy.
3. Histone deacetylase inhibitors include sodium butyrate, valproic acid, phenylbutyrate / VP-101 / EL-532, Pivanex / AN-9, OSU-HDAC42, trichostatin A, suberoylanilide hydroxamic acid (Zolinza / Volinostat / SAHA) ), LAQ-824, Panobinostat / LBH-589, CRA-24781 / PCI24781, Belinostat / PXD101, Tubacin, Oxamflatin, ITF2357, SB939, R306465 / JNJ-16241199, MS-275 / SNDXst-275 / G Pimelic diphenylamide, Romidepsin / FK228 / De sieptide, Apicidin, Trapoxin A, HC-Toxin, NCH-51, Alpha-mercaptoketone 19y / KD5150, 5- (Trifluoroacetyl) thiophene-2-Citropide, 2-Triboxamide, 2-Triboxamide The cancer therapeutic agent according to claim 1 or 2, which is selected from ketones.
4. An inhibitor of interstrand cross-link repair or nucleotide excision repair comprising a histone deacetylase inhibitor as an active ingredient.
5. A method of treating cancer by photochemotherapy, comprising administering a histone deacetylase inhibitor and a photosensitizer to a patient and then irradiating the site to be treated with UVA.
6. 6. The method of claim 5, wherein the photosensitizer is psoralen.
7. A method of inhibiting interstrand cross-linking repair or nucleotide excision repair caused by UVA irradiation in a patient undergoing cancer photochemotherapy, comprising administering to the patient a histone deacetylase inhibitor.
8. The method according to claim 7, wherein the cancer photochemotherapy is PUVA therapy.
9. Histone deacetylase inhibitors include sodium butyrate, valproic acid, phenylbutyrate / VP-101 / EL-532, Pivanex / AN-9, OSU-HDAC42, trichostatin A, suberoylanilide hydroxamic acid (Zolinza / Volinostat / SAHA) ), LAQ-824, Panobinostat / LBH-589, CRA-24781 / PCI24781, Belinostat / PXD101, Tubacin, Oxamflatin, ITF2357, SB939, R306465 / JNJ-16241199, MS-275 / SNDXst-275 / G Pimelic diphenylamide, Romidepsin / FK228 / De sieptide, Apicidin, Trapoxin A, HC-Toxin, NCH-51, Alpha-mercaptoketone 19y / KD5150, 5- (Trifluoroacetyl) thiophene-2-Citropide, 2-Triboxamide, 2-Triboxamide 9. A method according to any of claims 5-8, selected from ketones.

Images