Patent Application
20130310448
This application claims benefit under 35 U.S.C. §119 of Taiwanese Patent Application No. 101117920, filed on May 18, 2012, the contents of which are incorporated by reference as if fully set forth herein in its entirety.
The present invention relates to benzopyran-4-one derivatives compound capable of inhibiting ATR and FANCD2 activation, and more particularly to those benzopyran-4-one derivatives compound capable of improving the cancer sensitivity and poor prognosis to DNA-damaging therapeutics.
The benzopyran-4-one derivatives compound of formula I having a flavonoid moiety, which previously isolated and identified from the whole plant extract of Thelypteris torresiana, a fern species native to Taiwan.
The exposure and biological effects of Protoapigenone (I-1), 5′,6′-Dihydro-6′-methoxy-protoapigenone (I-2) and Protoapigenin (I-3) compounds have been investigated by cytotoxicity assay. It was found Protoapigenone (I-1) demonstrated therapeutic effects and was a lead compound for potential anticancer drug development.
Furthermore, for developing a new potent anticancer drug, several analogues of I type compound such as I-4, I-5, I-6, I-7 and I-8, and another II type moiety such as II-1 and II-2 compound are also synthesized or semi-synthesized. Where the compound I-4 having chemical name 2-(1-hydroxy-4-oxocyclohexa-2,5-dienyl)-4H-chromen-4-one can be expressed by the general names of protoflavonone. The compound I-5 is also termed as 5-hydroxyprotoflavone, whose chemical name is 2-(1-hydroxy-4-oxocyclohexa-2,5-dienyl)-5-hydroxy-4H-chromen-4-one. The compound I-6 having chemical name 5-hydroxy-2-(1-hydroxy-4-oxocyclohexa-2,5-dienyl)-7-methoxy-4H-chromen-4-one can be expressed by the general names of 5-hydroxy-7-methoxy-protoflavonone. The homologous compounds I-4 and 1-7 have the similar structure, but a different function group on R11 positions only, which is a hydroxyl group and another is methoxyl group in that position. The compound I-5, I-8, II-1 and II-2 also present the modified function group of R11 positions models.
Formula II having an β-naphthoflavone moiety, the compound II-1 has chemical name 3-(1-hydroxy-4-oxo-cyclohexa-2,5-dienyl)-1H-benzo[f]chromen-1-one. The compound II-2 has chemical name 1′-methoxy-β-naphthoflavone.
In previous studies, Protoapigenone (I-1) and its more potent analog compound II-1 (
In accordance with an aspect of the present invention, compounds of benzopyran-4-one derivatives, characteristically with inhibiting of DNA Damage Response (DDR), are provided. The benzopyran-4-one derivatives includes a common structure being the following formula I:
wherein: R3, R5, R7, R11, R14 and R16 are selected independently from a group consisting of a hydrogen, a hydroxyl group, a methoxyl group and a oxygen atom contain a double bond.
In accordance with a further aspect of the present invention, compounds of benzopyran-4-one derivatives, characteristically with ATR-mediated DNA damage checkpoint, is provided. The benzopyran-4-one derivatives includes a common structure being the following formula II;
wherein: R21 is selected independently from a group consisting of a hydrogen, a hydroxyl group and a methoxyl group.
In a further aspect of the present invention, a method for assaying a state of DNA DDR kinase signaling cascades is provided. The method includes steps of:
The above objects and advantages of the present aspects will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings, in which:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee
a)-3(b) show that dose-dependent effects of compound I-1 and compound II-1 on the inhibition of UV-induced Chk1 phosphorylation in cells.
a) illustrates immunoblots showing the expression of MDA-MB-231 cell.
b) illustrates immunoblots showing the expression of A549 cell.
a)-5(b) show benzopyran-4-one derivatives inhibit DNA damage-induced DDR.
a) illustrates immunoblots showing the expression of inhibit Chk1 phosphorylation.
b) illustrates immunoblots showing the expression of A549 cell. Cells pretreated with 50 μM okadaic acid (OA) or 20 μM MG132 and subjected to 10 J/m2 UV for 1 h to induce DDR.
a)-6(b) show benzopyran-4-one derivatives inhibit UV-induced Chk1 phosphorylation.
a) illustrates immunoblots showing the expression of A549 cell.
b) illustrates immunoblots showing the expression of MDA-MB-231 cell. Cells pretreated with chemicals for 20 min and subjected to 10 J/m2 UV for 1 h to induce DDR.
a)-8(b) show benzopyran-4-one derivatives inhibit ATR-dependent Chk1 phosphorylation.
a) illustrates immunoblots showing the expression DDR induced by H2O2 (peroxide).
b) illustrates immunoblots showing the expression DDR induced by hydroxyurea (HU).
a)-10(c) show benzopyran-4-one derivatives inhibit UV- or H2O2-induced Chk1 phosphorylation
a) illustrate immunoblots showing compound I-1 inhibit H2O2-induced Chk1 phosphorylation.
b) illustrate immunoblots showing compound I-1 inhibit UV-induced Chk1 phosphorylation.
c) illustrate immunoblots showing compound II-1 inhibit H2O2-induced Chk1 phosphorylation.
a)-12(b) show benzopyran-4-one derivatives inhibit DNA damage checkpoint and repair.
a) illustrates percentages of the mitotic marker.
b) illustrates the expression FACS (Fluorescence Activated Cell Sorting) dot blot for analyzing the percentage of GFP cell denoting the HRR frequency.
a)-14(c) show the benzopyran-4-one derivatives inhibit cisplatin-induced Chk1 phosphorylation and FANCD2 monoubiquitination.
a) illustrates immunoblots showing the effect on cisplatin-induced DDR in A594 cell.
b) illustrates immunoblots showing the effect on cisplatin-induced DDR in U2OS cell.
c) illustrates immunoblots showing the effect on cisplatin-induced DDR in MDA-MB-231 cell.
a)-15(b) show that in vitro clonogenic survival for A549 cell.
a) illustrates the effects fraction of compound I-1.
b) illustrates the effects fraction of compound II-1.
a)-16(b) show that in vitro clonogenic survival for MDA-MB-231 cell.
a) illustrates the effects fraction of compound I-1.
b) illustrates the effects fraction of compound II-1.
a)-17(b) show chemosensitization effect of benzopyran-4-one derivatives.
a) illustrates the effects fraction of compound I-1.
b) illustrates the effects fraction of compound II-1.
a)-19(b) show that optical activity of benzopyran-4-one derivatives.
a) illustrates the effects on A549 cells
b) illustrates the effects on MDA-MB-231 cells
a)-20(b) show that optical activity affected by benzopyran-4-one derivatives concentration.
a) illustrates the effects of compound I-1.
b) illustrates the effects of compound II-1.
a)-21(b) show that rate of cell cycle progression.
a) illustrates the effects of unsynchronized cells.
b) illustrates the effects of control group.
a)-22(c) show that rate of cell cycle progression were treated with benzopyran-4-one derivatives for 6 hrs.
a) illustrates the effects of control group.
b) illustrates the effects of compound I-1.
c) illustrates the effects of compound II-1.
a)-23(c) shows that rate of cell cycle progression were treated with benzopyran-4-one derivatives for 9 hrs.
a) illustrates the effects of control group.
b) illustrates the effects of compound I-1.
c) illustrates the effects of compound II-1.
a)-24(e) show that number of labeled DNA replication.
a) illustrates the effects of DMSO group.
b) illustrates the effects of Hydroxyurea (HU) group.
c) illustrates the effects of ku55933 group.
d) illustrates the effects of compound I-1.
e) illustrates the effects of compound II-1.
Further embodiments herein may be formed by supplementing an embodiment with one or more element from any one or more other embodiment herein, and/or substituting one or more element from one embodiment with one or more element from one or more other embodiment herein.
The following non-limiting examples are provided to illustrate particular embodiments. The embodiments throughout may be supplemented with one or more detail from one or more example below, and/or one or more element from an embodiment may be substituted with one or more detail from one or more example below.
Ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) are 2 members of the phosphoinositide 3-kinase (PI3K)-related protein kinases family that play a central role in DNA damage response (DDR) coordination; they also function in the signaling cascades machinery of cell cycle arrest, DNA repair and transcription, and cell death. While ATM is predominant activated in response to DNA strand breaks, ATR is activated in response to damage arising from ultraviolet (UV) ray or replication block; both kinases activate signaling cascades that involving 2 checkpoint kinases effectors, Chk1 and Chk2, whose roles were previously suggested to be redundant. In contrast to ATM, ATR has been reported to be indispensible for cell growth and for life. ATR-knockout mouse embryos died early due to mitotic catastrophe characterized by incomplete DNA replication and chromosomal fragmentation. Moreover, ATR gene mutations are rarely found in humans. The only mutated variants that can survive are heterozygous or hypomorphic variants. Furthermore, cells derived from patients with Seckel syndrome exhibit cellular features associated with ATR signaling cascades defects. Consistent with this phenotype, seckel-like mouse embryonic cells showed accelerated aging due to replicative stress, exhibiting an accumulation of lethal chromosomal breaks. However, with regard to its role in regulating the replication checkpoint, ATR is activated by most cancer chemotherapeutic agents that target DNA in replicating cells. Therefore, inhibition of ATR signaling cascades is a valid and promising strategy that can improve chemotherapeutic or radiotherapeutic efficiency.
Thus so far, several inhibitors of DDR-related kinases, including Chk1 and Chk2, have been successfully used alone or in combination with each other in clinical trials. Recently, several chemicals that inhibit ATR kinase activity in vitro were used to support the hypothesis that ATR kinase can be targeted to improve cancer therapy. Since most of these studies are in their initial stages, it is imperative to focus more efforts toward investigating strategies to inhibit ATR signaling cascades.
In accordance with an aspect of the present invention, benzopyran-4-one derivatives compound, characteristically with inhibiting of DNA Damage Response (DDR), is provided.
Another aspect of this invention, pharmaceutical composition of benzopyran-4-one derivatives, characteristically with inhibiting of DNA Damage Response (DDR), is provided. The benzopyran-4-one derivatives includes a common structure being the following formula I or formula II,
In a further aspect of this invention is directed towards a method of treating cancer in a subject in need thereof, including the sequential or simultaneous co-administration of a compound of benzopyran-4-one derivatives or a pharmaceutically acceptable salt thereof, and a DNA-damaging agents. In some embodiments, the DNA-damaging agents are selected from chemotherapeutic drugs such as alkylating agents, antimetabolic agents, antibiotic anti-cancer agents, Topoisomerase inhibitors and anti-mitosis agents.
In some embodiments, the alkylating agent is one selected from Nitrogen mustards (eg. Melphalan, mechlorethamine, Chlorambucil, Ifosfamide, Cyclophosphamide, Estramustine and phenoxybenzamine); or Aziridines (eg. Thiotepa, Carboquone); or Nitrosoureas (eg. Carmustine, Semustine, Iomustine, Nimustine, Streptozocin, Ranimustine and Lomustine); or Procarbazine and triazenes (eg. Dacarbazine, Temozolomide and Procarbazine); or Alkyl sulfonate (eg. Busulfan); or Platinum coordination complex (eg. Cisplatin, Carboplatin, Nedaplatin, Iproplatin and Oxaliplatin); and mixtures thereof.
In some embodiments, the antimetabolic agent is one selected from Thymidylate synthase inhibitor (eg. Aminopterin, Methotrexate, Tegafur, Piritrexin, Trimetrexate, Floxuridine, Raltitrexed, Pemetrexed, Fluorouracil, Doxifluridine and Capecitabine); or Amidophosphoribosyl transferase inhibitors (eg. Mercaptopurine, Thioguanine and Thionosine); or DNA chain elongation inhibitors (eg. Cytarabine, Ancitabine, Gemcitabine, Fludarabine, Cladribine, Clofarabine, Azaserine, Azacitidine, Pentostatin, Hydroxyurea); and mixtures thereof.
In some embodiments, the antibiotic anti-cancer agent is one selected from free radical agents (eg. Bleomycin and Actinomycin D); or Topoisomerase II inhibitors (eg. Daunorubicin, Doxorubicin, Idarubicin, Epirubicin, valrubicin, Pirarubicin, Aclarubicin, Mitoxantrone and Piroxanthrone); or other therapies or anticancer agents (eg. Menogaril, Plicamycin, Acivicin, Anthramycin, Pentostatin, Calicheamicin and Peplomycin); and mixtures thereof.
In some embodiments, the Topoisomerase inhibitor is one selected from Topoisomerase I inhibitors (eg. Camptothecin, Irinotecan, Topotecan); or Topoisomerase II (eg. Podophyllin, Podophyllotoxin, Etoposide, Teniposide); and mixtures thereof.
In some embodiments, the anti-mitosis agent is one selected from Paclitaxel and Docetaxel; or anti-microtubule agents (eg. Colchicine, Vinblastine, Vincristine, Vindesine and Vinorelbine); and mixtures thereof.
Benzopyran-4-one derivatives compounds of this invention include Protoapigenone (I-1), 5′,6′-dihydro-6′-methoxy-protoapigenone (I-2), Protoapigenin, (I-3), protoflavonone (I-4), 5-hydroxyprotoflavone (I-5), 5-hydroxy-7-methoxy-protoflavonone (I-6), compounds I-7, compounds I-8, 3-(1-hydroxy-4-oxocyclohexa-2,5-dienyl)-1-H-benzo[f]chromen-1-one (II-1) and compounds II-2.
In a further aspect of this invention, pharmaceutical composition of benzopyran-4-one derivatives, characteristically with modulating the activation state of ATM kinase is provided. The benzopyran-4-one derivatives includes a common structure being the following formula I or formula II,
In a further aspect of this invention, both assay kit and assay composition of benzopyran-4-one derivatives, characteristically with detecting the activation state of ATR, DNA DDR kinase signaling cascades is provided.
In a further aspect of this invention is directed towards a method of analyzeing ATR, DNA DDR kinase signaling cascades in a reaction site thereof, including the sequential or simultaneous of benzopyran-4-one derivatives compound or a pharmaceutically acceptable salt thereof, and a chemotherapeutic drugs or additional agents. In some embodiments, the chemotherapeutic drug is selected from chemotherapeutic drugs such as alkylating agents, antimetabolic agents, antibiotic anti-cancer agents, Topoisomerase inhibitors and anti-mitosis agents.
In some embodiments, the individual components of the combination may be administered separately, at different times during the course of therapy, or concurrently, in divided or single combination forms. Also provided is, for example, simultaneous, staggered, or alternating treatment.
In a further aspect of this invention, compounds and pharmaceutical composition of benzopyran-4-one derivatives, characteristically with detecting of DNA damage in cancer cell as determined by the activation state of ATM kinase is also useful for monitoring therapeutic effects during treatment.
In some embodiments, method using benzopyran-4-one derivatives compound or pharmaceutical composition for defecting in the ATR signaling cascade and/or DNA-damage response (DDR). In some embodiments, the defect is altered expression or activity of one or more of the following cell markers as determined by standard cell marker detection assays: ATM, CHK1, CHK2, cellular tumor antigen p53, Adenosine monophosphate activated protein kinase (AMPK), mammalian target of rapamycin complex (mTORC) 1, metal response element (MRE) 11, mitogen-activated protein kinase (MAPK), MAPK-activated protein kinase (MAPKAPK) 2, DNA Repair Protein (RAD50), Nijmegen breakage syndrome (NBS) 1, 53BP1, mediator of DNA damage checkpoint (MDC) 1, H2A histone family member X (H2AX).
In another embodiment, the cell is a cancer cell expressing DNA damaging oncogenes. In some embodiments, the cancer cell has altered expression or activity of one or more of the following cell markers as determined by standard cell marker detection assays: K-Ras, N-Ras, H-Ras, Raf, Myc, Mos, E2F, Cdc25A, CDC4, CDK2, Cyclin E, Cyclin A and Rb.
In a further embodiment, the invention relates to an assay kit or assay composition for determent of ATR and/or DNA DDR signaling cascades at reaction site. In particular the assay kit or assay composition can include, a benzopyran-4-one derivatives compound, a processing/handling plan, a compartment, a additional reagent and instructions for use, or a reagent with a compartment and instructions for use. In one embodiment, for the purpose of altered expression or activity can then generate a detectable at the reaction site of the immunocomplex.
The additional reagent can include ATR, the ATR receptor, the complex of DNA, or an antigenic fragment thereof, a binding composition, or a nucleic acid. A kit for determining the binding of a test compound, e.g., acquired from a biological sample or from a chemical library, can include a control compound, a labeled compound, and a method for separating free labeled compound from bound labeled compound and a combination thereof.
The term excipients or “pharmaceutically acceptable carrier or excipients” and “bio-available carriers or excipients” above-mentioned include any appropriate compounds known to be used for preparing the dosage form, such as the solvent, the dispersing agent, the coating, the anti-bacterial or anti-fungal agent and the preserving agent or the delayed absorbent. Usually, such kind of carrier or excipient does not have the therapeutic activity itself. Each formulation prepared by combining the derivatives disclosed in the present invention and the pharmaceutically acceptable carriers or excipients will not cause the undesired effect, allergy or other inappropriate effects while being administered to human. Accordingly, the derivatives disclosed in the present invention in combination with the pharmaceutically acceptable carrier or excipients are adaptable in the clinical usage and in the human. A therapeutic effect can be achieved by using the dosage form in the present invention by the local or sublingual administration via the venous, oral, and inhalation routes or via the nasal, rectal and vaginal routes. About 0.1 mg to 1000 mg per day of the active ingredient is administered for the patients of various diseases.
The carrier is varied with each formulation, and the sterile injection composition can be dissolved or suspended in the non-toxic intravenous injection diluents or solvent such as 1,3-butanediol. Among these carriers, the acceptable carrier may be mannitol or water. Besides, the fixing oil or the synthetic glycerol ester or di-glycerol ester is the commonly used solvent. The fatty acid such as the oleic acid, the olive oil or the castor oil and the glycerol ester derivatives thereof, especially the oxy-acetylated type, may serve as the oil for preparing the injection and as the naturally pharmaceutical acceptable oil. Such oil solution or suspension may include the long chain alcohol diluents or the dispersing agent, the carboxylmethyl cellulose or the analogous dispersing agent. Other carriers are common surfactant such as Tween and Spans or other analogous emulsion, or the pharmaceutically acceptable solid, liquid or other bio-available enhancing agent used for developing the formulation that used in the pharmaceutical industry.
The composition for oral administration adopts any oral acceptable formulation, which includes capsule, tablet, pill, emulsion, aqueous suspension, dispersing agent and solvent. The carrier generally used in the oral formulation, taking a tablet as an example, the carrier may be lactose, corn starch and lubricant, and magnesium stearate is the basic additive. The excipients used in a capsule include lactose and dried corn starch. For preparing an aqueous suspension or an emulsion formulation, the active ingredient is suspended or dissolved in oil interface in combination with the emulsion or the suspending agent, and appropriate amount of sweetening agent, flavors or pigment is added as needed.
The nasal aerosol or inhalation composition may be prepared according to the well-known preparation techniques. For example, the bioavailability can be increased by dissolving the composition in the phosphate buffer saline and adding the benzyl alcohol or other appropriate preservative, or the absorption enhancing agent. The compound of the present invention may be formulated as suppositories for rectal or virginal administration.
The compound of the present invention can also be administered intravenously, as well as subcutaneously, parentally, muscular, or by the intra-articular, intracranial, intra-articular fluid and intra-spinal injections, the aortic injection, the sterna injection, the intra-lesion injection or other appropriate administrations.
Protoapigenone (I-1) induces chromosomal aberrations but does not produce marked DDR.
Previously, Protoapigenone (I-1) and compound II-1 were demonstrated to cause DNA strand breaks and apoptosis in lung and prostate cancers (Chen H M, et al., Free Radic Biol Med 2011), suggesting that inducing DNA damage may be the potential mechanism underlying the anticancer effect of benzopyran-4-one derivatives.
To test this hypothesis, it is investigated the cytogenetic effect of Protoapigenone (I-1) on CHO cells (
Since mitomycin C can induce DDR in many cancers, it is investigated what kind of DDR signaling was activated by Protoapigenone (I-1). Surprisingly, high doses of Protoapigenone (I-1) in HEK293T cells did not induce noticeable changes in the putative DDR signaling, which it is measured by analyzing the phosphorylation of the ATM-dependent Chk2 Thr68 residue and the ATR-dependent Chk1 Ser345 residue (
Protoapigenone (I-1) and compound II-1 inhibit Chk1 phosphorylation after DNA damage.
Understanding the mechanism by which the benzopyran-4-one derivatives compounds cause chromosomal breakages (
It is speculated that the replacement of 2 hydroxyl groups on Protoapigenone (I-1) with an additional benzene ring contributes positively to this ATR inhibition; however, the definite pharmacophores need to be further investigated when the ATR protein structure is resolved.
Target specificity of Protoapigenone (I-1) and compound II-1 for ATR-mediated signaling inhibition.
To elucidate the specificity of the benzopyran-4-one derivatives inhibition on ATR-mediated signaling, it is compared the change between cells treated with benzopyran-4-one derivatives or the ATM-specific inhibitor KU55933 before the induction of DDR. After H2O2 damage, ATM is thought to be the principal responder, and KU55933 treatment strongly inhibited ATM-mediated Chk2 phosphorylation specifically, but its effect on ATR-mediated Chk1 phosphorylation was small (
Using these pharmacological methods, it is demonstrated that the specificity of DDR inhibition between benzopyran-4-one derivatives and KU55933 was completely different. To strengthen the argument that benzopyran-4-one derivatives specifically inhibits ATR signaling, small inhibitory RNAs against ATM, ATR, and the catalytic subunit of DNA protein kinase (DNA-PKcs) were introduced into HEK293T cells before exposure to UV or H2O2.
Our results demonstrated that benzopyran-4-one derivatives completely inhibited UV-induced or H2O2-induced Chk1 phosphorylation in a manner similar to siRNA knockdown of ATR, but not ATM or DNA-PKcs (
To further identify the specific mediator that contributes to the effect of Protoapigenone (I-1) on the initiation of ATR kinase activation, it is tested whether TopBp1, ATRIP, and claspin were involved, as they have been identified as mediators of ATR kinase activation (Lopez-Contreras A J, et al. DNA Repair (Amst) 2010). Our results demonstrated that overexpression of ATRIP or TopBP1 did not reverse the inhibitory effect of Protoapigenone (I-1) on Chk1 phosphorylation, whereas overexpression of claspin or ATR did (
Protoapigenone (I-1) and compound II-1 impair the functions of DNA damage checkpoints and DNA repair.
Previously, it has been demonstrated that S/M and G2/M checkpoints are activated by ATR in response to different types of DNA damage (Nghiem P, et al. Proc Natl Acad Sci USA 2001). Of these, the G2/M checkpoint involves ATM and ATR in collaboration, whereas the S/M checkpoint is mediated solely by ATR. To maintain genetic integrity, ATR can prevent premature mitotic entry in the event of incomplete DNA replication or unrepaired DNA damage. In order to evaluate the effect of benzopyran-4-one derivatives on these ATR-associated DNA damage checkpoints, it is observed the effect of benzopyran-4-one derivatives on mitotic entry following hydroxyurea or cisplatin treatment. In MDA-MB-231 cells, hydroxyurea and cisplatin significantly decreased the number of mitotic cells, indicating that the S/M and G2/M checkpoints are intact in MDA-MB-231 cells (
Benzopyran-4-one derivatives or KU55933 treatment increased the percentage of mitotic cells in cisplatin-treated cells, as the Table 2 suggesting that all of these compounds inhibited the damage-induced G2/M checkpoint. However, benzopyran-4-one derivatives, but not KU55933, significantly increased the HU-induced mitotic entry that is specific for the S/M checkpoint, indicating that benzopyran-4-one derivatives specifically impaired this distinctive checkpoint mediated solely by ATR (
ATR function is also linked to DNA repair via its coupled targets (Sorensen C S, et al. Nat Cell Biol 2005). To examine the effect of benzopyran-4-one derivatives treatment on DNA repair, it is performed a homologous recombination repair (HRR) assay in HeLa cells.
Our result, as Table 3 demonstrated that chromosomal breaks normally repaired by HRR were dose-dependently inhibited by Protoapigenone (I-1) at low concentrations. Compound II-1 produced similar effects at doses that were 10-fold lower than that of Protoapigenone (I-1) (
Protoapigenone (I-1) and compound II-1 enhance chemosensitivity.
Inhibition of the checkpoint and repair mechanisms leads to chemosensitization in cancers. It is questioned whether benzopyran-4-one derivatives could function as sensitizers for the chemotherapeutic drugs cisplatin that has been shown to induce ATR activation as well as FANCD2 monoubiquitination, which is the vital step for DNA crosslink repair (Chirnomas D, et al. Mol Cancer Ther 2006). It is found that benzopyran-4-one derivatives treatment decreased the cisplatin-induced Chk1 phosphorylation and FANCD2 monoubiquitination in A549, MDA-MB-231, and U2OS cells (
ATR are involved in DNA replication. Low doses of Protoapigenone (I-1) and compound II-1 significantly slowed cancer growth in a dose-dependent manner (
Through EdU (5-ethynyl-2′-deoxyuridine) incorporation to measurement the capacity of DNA replication, 4 μM protoapigenone (I-1) (
Materials and Methods
Antibodies
Primary antibodies of Chk1 (sc-8408), Chk2 (sc-17747), FANCD2 (SC-20022), phospho-ATM Ser1981 (sc-47739), and Myc (sc-40) were purchased from Santa Cruz. Phospho-histone H3 Ser10 (06-570) and H2AX Ser139 (05-636) antibodies were purchased from Millipore. Claspin (2880) and phospho-Chk1 Ser345 (2348), phospho-Chk2 Thr68 (2661), phospho-P53 Ser15 (9286), P38 MAPK Thr180/Tyr182 (9216), and MAPKAPK2 Thr334 (3007) were purchased from Cell Signaling. Actin (A2066), flag (F 1804), and hemagglutinin (H9658) antibodies were purchased from Sigma-Aldrich. ATR (A300-137A), ATRIP (A300-095A), and TopBP1 (A300-111A) antibodies were purchased from Bethyl; and anti-ATM (GTX70103) antibodies were purchased from Gene Tex.
Cell Culture and Treatment
MDA-MB-231 (breast adenocarcinoma; ATCC HTB-26, BCRC 60425) and A549 (lung adenocarcinoma; ATCC CCL-185, BCRC 60074) human cell lines were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan), and were authenticated by American Type Culture Collection (ATCC, Manassas, Va.). U2OS (osteosarcoma), HeLa (cervical adenocarcinoma), and HEK 293T (embryonic kidney cells) human cell lines were provided by Dr. Sheau-Yann Shieh (Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Gibco). For DDR induction, freshly diluted H2O2 (Merck) was added to the culture medium 1 h before the cells were harvested. For UV irradiation treatment, the cells were irradiated for 10 J/m2 by a cross-linker (UVP) 1 h prior to analysis. Protoapigenone (I-1) and compound II-1 were isolated and synthesized as described previously (15-17).
In Vitro Chemosensitization Assay
To evaluate in vitro chemosensitization, cells were seeded in 6-well plates 1 d before the experiment at a density of 100-400 cells/well. The drugs were incubated with the cells for 6 h, after which the medium was replaced with fresh drug-free FBS-containing medium. The colonies became visible and were counted 7-10 d later using 0.1% crystal violet staining following image capture by a CCD camera (LAS-4000 mini; Fujifilm).
Flow Cytometry
To evaluate the effect of DNA damage checkpoint activation on cell cycle distribution, the cells were harvested at indicated time points and fixed with methanol for at least 2 h. The DNA was then stained with a solution containing propidium iodide (PI) and RNase A (Sigma-Aldrich). Fluorescently labeled cells were subsequently analyzed by the flow cytometer (LSR II; BD Biosciences) with a suitable selection of excitation and emission wavelengths. The percentages of different fluorescent cell populations were analyzed using WinMDI Ver. 2.9 (The Scripps Research Institute).
DNA replication was measured using a Click-it EdU assay kit, which is based on incorporation of the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) into DNA during replication (Invitrogen). Then, 10 μM EdU was added to the cell culture medium 30 min before the cells were harvested and fixed in 4% paraformaldehyde. After cycloaddition, EdU was detected with Alexa Fluor 647 using click reaction catalyzed by Cu (II), and the DNA content was stained by CellCycle 405-blue. To assay the mitotic entry, cells were treated with the indicated drugs and trapped in 70 nM nocodazole for 16 h, and antibodies against phospho-histone H3 Ser10 and PI were used to stain the mitotic cells and DNA content, respectively. An FITC Annexin V apoptosis detection kit was used to characterize the phenotype of cell death based on PI and Annexin V double staining (BD Pharmingen, San Diego, Calif.). Fluorescence-labeled cells were subsequently analyzed by the BD LSR II flow cytometer with a suitable selection of excitation and emission wavelengths. The percentages of different fluorescent cells were analyzed using WinMDI Ver. 2.9.
In Vitro Chromosome Aberration Test
In brief, 5×105 Chinese Hamster Ovary (CHO) cells were seeded in 60-mm dishes 1 d before the experiment. Protoapigenone (I-1)-induced structural chromosomal changes after 20 h were compared with that of the cells cultured in 2 μM mitomycin C. At 18 h after Protoapigenone (I-1), 0.1 μg/mL colchicine was added for 2 h, and metaphase cells were collected by shaking them off the dishes. Mitotic cells were treated with 0.5% KCl for 10 min and fixed with a 3:1 mixture of methanol: glacial acetic acid. The cells were then spread on slides for chromosome staining with 5% Giemsa solution. It is then analyzed the chromosome structure of 200 well-spread metaphase cells (100 metaphase cells/experiment) under a 100× oil immersion objective.
Plasmids and siRNAs
The plasmids ATR, ATRIP, and claspin were kindly provided by Dr. X. Wu (The Scripps Research Institute, La Jolla, Calif.), and TopBP1 was provided by Dr. J. Chen (University of Texas MD Anderson Cancer Center, Houston, Tex.). The siRNA sequences of the target ATM (5′-AAGCGCCTGATTCGAGATCCT-3′), ATR (5′-CCTCCGTGATGTTGCTTGATT-3′), DNA-PKcs (5′-GATCGCACCTTACTCTGTTGA-3′), and the random sequence that served as the control (5′-AAGTCAATATGCGACTGATGG-3′) were synthesized by Sigma-Proligo (23,24). All transfections in HEK293T cells were performed by the calcium phosphate precipitation method.
Western Immunoblotting.
Cell lysate preparations, gel electrophoresis, and immunoblotting were performed as previously described (23). The binding of primary antibodies were detected by horseradish peroxidase-coupled secondary antibodies (Jackson ImmunoResearch) followed by enhanced chemiluminescence (ECL)(Millipore). The images of non-saturated bands were captured using a luminescent image analyzer (LAS-4000 mini; Fujifilm). The antibodies used in this study are listed in supplementary materials.
DNA Homologous Recombination Repair Assay.
DNA constructs of the recombination substrate pHPRT-DRGFP, in which the I-SceI site lies within 1 copy of 2 mutated tandem repeated GFP genes, and the I-SceI endonuclease expression vector pCBASceI, were originally constructed by Dr. M. Jasin (25). In brief, it is generated a stable pHPRT-DRGFP construct in HeLa cells, and evaluated the chromosomal breaks generated by I-SceI endonuclease expression. Six hours after pCBASceI was delivered into the cells, complete medium with or without Protoapigenone (I-1) or compound II-1 was replaced onto the cells. Forty-eight hours after delivery, the efficiency of chromosomal HRR was obtained as the percentage of GFP-positive cells, which was assessed by flow cytometry.
Human Xenograft Tumors in Nude Mice.
Human breast cancer MAD-MB-231 cells were harvested from the culture, resuspended in medium without serum at 1×108 cells/mL, and 0.1 mL of this suspension was subcutaneously injected into the right flank of female nude mice (BALB/cAnN-Foxn1nu/Crl Narl; purchased from the National Science Council Animal Center, Taiwan). Tumor-injected mice that successfully developed tumors that grew to approximately 50-100 mm3 in volume were randomly sorted into groups and used for the experiments. Control vehicle or 2 mg/kg of cisplatin with or without 0.2 mg/kg of compound II-1 was administered intraperitoneally every 4 d throughout the experiment.
Tablets are prepared using standard mixing and formation techniques as described in U.S. Pat. No. 5,358,941, to Bechard et al., issued Oct. 25, 1994, which is incorporated by reference herein in its entirety.
A inhibiting of DNA Damage Response composition including a benzopyran-4-one derivatives compound presented by formula I:
A inhibiting of DNA Damage Response composition including a benzopyran-4-one derivatives compound presented by formula II:
A medical effect for inhibited ATR-mediated DNA damage checkpoint composition including an effective amount of being one compound selected from a group consisting of benzopyran-4-one derivatives represented by formula I and formula II.
A sensitizing effect for chemotherapeutic treatment composition including an effective amount of a compound selected from a group consisting of benzopyran-4-one derivatives represented by formula I and formula II.
A assay composition for providing assay for the state of DNA DDR signaling cascade, including an effective amount of a compound selected from a group consisting of benzopyran-4-one derivatives represented by formula I and formula II.
A pharmaceutical as above embodiments, therewith in a subject in need thereof including co-administration a compound of benzopyran-4-one derivatives; and at least one chemotherapeutic drugs against a cancer disease in need thereof.
A pharmaceutical as above embodiments, therewith the chemotherapeutic drugs includes one selected from an alkylating agent, an antimetabolic agents, an antibiotic anti-cancer agents, a Topoisomerase I, a Topoisomerase II, an anti-mitosis agents and a combination thereof.
A pharmaceutical as above embodiments, therewith the alkylating agent is one selected from Nitrogen mustards (eg. Melphalan, mechlorethamine, Chlorambucil, Ifosfamide, Cyclophosphamide, Estramustine and phenoxybenzamine); or Aziridines (eg. Thiotepa, Carboquone); or Nitrosoureas (eg. Carmustine, Semustine, Iomustine, Nimustine, Streptozocin, Ranimustine and Lomustine); or Procarbazine and triazenes (eg. Dacarbazine, Temozolomide and Procarbazine); or Alkyl sulfonate (eg. Busulfan); or Platinum coordination complex (eg. Cisplatin, Carboplatin, Nedaplatin, Iproplatin and Oxaliplatin); and a combination thereof.
A pharmaceutical as above embodiments, therewith the antimetabolic agent is one selected from Thymidylate synthase inhibitor (eg. Aminopterin, Methotrexate, Tegafur, Piritrexin, Trimetrexate, Floxuridine, Raltitrexed, Pemetrexed, Fluorouracil, Doxifluridine and Capecitabine); or Amidophosphoribosyl transferase inhibitors (eg. Mercaptopurine, Thioguanine and Thionosine); or DNA chain elongation inhibitors (eg. Cytarabine, Ancitabine, Gemcitabine, Fludarabine, Cladribine, Clofarabine, Azaserine, Azacitidine, Pentostatin, Hydroxyurea); and a combination thereof.
A pharmaceutical as above embodiments, therewith the antibiotic anti-cancer agent is one selected from free radical agents (eg. Bleomycin and Actinomycin D); or Topoisomerase II inhibitors (eg. Daunorubicin, Doxorubicin, Idarubicin, Epirubicin, valrubicin, Pirarubicin, Aclarubicin, Mitoxantrone and Piroxanthrone); or other therapies or anticancer agents (eg. Menogaril, Plicamycin, Acivicin, Anthramycin, Pentostatin, Calicheamicin and Peplomycin) and a combination thereof.
A pharmaceutical as above embodiments, therewith the Topoisomerase inhibitor is one selected from Topoisomerase I inhibitors (eg. Camptothecin, Irinotecan, Topotecan); or Topoisomerase II (eg. Podophyllin, Podophyllotoxin, Etoposide, Teniposide) and a combination thereof.
A pharmaceutical as above embodiments, therewith the anti-mitosis agent is one selected from Paclitaxel and Docetaxel; or anti-microtubule agents (eg. Colchicine, Vinblastine, Vincristine, Vindesine and Vinorelbine) and a combination thereof.
A method for sensitizing cells to DNA damaging agents, including steps of providing an effective amount of a benzopyran-4-one derivative; and administering the effective amount of the benzopyran-4-one derivative to a subject in need thereof.
A method for inhibiting a DNA damage response (DDR), including steps of providing an effective amount of a benzopyran-4-one derivative; and administering the effective amount of the benzopyran-4-one derivative to a subject in need thereof.
A method as above embodiments, wherein the benzopyran-4-one derivative is represented by formula I:
A method as above embodiments, wherein the benzopyran-4-one derivative is represented by formula II:
A method as above embodiments, wherein the administering step further includes a step of co-administering the benzopyran-4-one derivative and at least one of chemotherapeutic drug against a cancer disease to the subject in need thereof.
A method as above embodiments, wherein the chemotherapeutic drugs include one selected from a group consisting of an alkylating agent, an antimetabolic agent, an antibiotic anti-cancer agent, a Topoisomerase I, a Topoisomerase II, an anti-mitosis agent and a combination thereof.
A method for inhibiting an ATR-mediated DNA damage checkpoint, including a step of administering to a subject in need thereof an effective amount of a compound being one of formula I and formula II,
| Number | Date | Country | Kind |
|---|---|---|---|
| 101117920 | May 2012 | TW | national |
