WO2010023917A1 - 抗がん剤の作用増強剤及びその利用、並びにがん患者の予後推定用バイオマーカー及びその利用 - Google Patents
抗がん剤の作用増強剤及びその利用、並びにがん患者の予後推定用バイオマーカー及びその利用 Download PDFInfo
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Definitions
- the present invention relates to a drug that enhances the action of an anticancer drug and its use, a biomarker for prognosis estimation of cancer patients, its use, and the like.
- Histone deacetylase controls cancer development by controlling cell growth, differentiation and survival (Non-patent Documents 1 and 2).
- HDACi inhibitors of HDACs
- Non-patent Documents 2 and 3 the molecular mechanism by which HDACi exerts a synergistic effect is not fully understood.
- Non-Patent Documents 2, 3, 4 Systematic in vitro studies have shown that HDACi specifically induces tumor cell growth arrest, differentiation, cell death, tumor angiogenesis inhibition, and the like.
- SAHA suberoylanilide hydroxamic acid
- FDA US Food and Drug Administration
- chemotherapeutic agents including cisplatin, 5-FU, etoposide, doxorubicin, etc. are known to induce the production of reactive oxygen species (ROS) as one of the cytotoxic mechanisms (Non-patent Document 6). ⁇ 9). It has been reported that changes in the expression of antioxidant enzymes or related genes regulate the resistance of cancer cells to chemotherapeutic agents (Non-Patent Documents 10 to 13). We support the finding that induction of ROS by therapeutic agents is important for cytotoxic activity.
- ROS reactive oxygen species
- Histone deacetylase inhibitors molecular mechanisms of action. Oncogene. 26 (37): 5541-52 (2007). Marks, PA. And Breslow, R. Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug. Nature Biotechnology. 25 (1): 84-90 (2007). Miyajima, A. et al. Role of reactive oxygen species in cis-dichlorodiammineplatinum-induced cytotoxicity on bladder cancer cells. Br J Cancer. 76 (2): 206-10 (1997). Kurosu, T., Fukuda, T., Miki, T. and Miura, O.
- BCL6 overexpression prevents increase in reactive oxygen species and inhibits apoptosis induced by chemotherapeutic reagents in B-cell lymphomaco44 68 (2003). Hwang, IT. Et al. Drug resistance to 5-FU linked to reactive oxygen species modulator 1. Biochem Biophys Res Commun. 359 (2): 304-10 (2007). Ravid, A. et al. 1,25-Dihydroxyvitamin D3 enhances the susceptibility of breast cancer cells to doxorubicin-induced oxidative damage. Cancer Res. 59 (4): 862-7 (1999). Godwin, AK. Et al. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis.
- anticancer drugs are often used in combination.
- the combined use of anticancer agents is performed for the purpose of improving the therapeutic effect, but sometimes causes serious side effects. This is because many of the currently used anticancer agents target fast-growing cells and exert cytotoxic effects on normal cells other than cancer cells.
- the present invention provides means for enhancing the action of anticancer agents, or provides a biomarker useful for understanding the prognosis of cancer patients and uses thereof, and results of cancer treatment. Contribute to the improvement of
- HDACi changes the expression of Thioredoxin and TBP-2 that control the oxidation state in cells.
- the present inventors have hypothesized that HDACi makes cancer cells sensitive to anticancer agents by controlling intracellular antioxidant mechanisms.
- (1) high expression in human tumors, (2) characteristics of controlling cell proliferation, differentiation, and survival (Halkidou K, Gaughan L, Cook S, Leung HY, Neal DE , Robson CN. (2004). Upregulation and nuclear recruitment of HDAC1 in hormone refractory prostate cancer. Prostate 59: 177-189 (Non-Patent Document 14); Glozak MA, Seto E. (2007).
- Histone deacetylases gene 26 HDAC1 with 5420-5432 (Non-Patent Document 15), (1) high expression in cancer cell lines, and (2) limited expression in vivo (Takahashi M, Inaguma Y) , Hiai H, Hirose F. (1988). Developmentally regulated expression of a human "finger" -containing gene encoded by the 5 'half of the ret transforming gene. Mol Cell Biol. 1: 1853-6 ; Tezel, G. et al. M. (1999). Different nuclear / cytoplasmic distributions of RET finger pr otein in different cell types. Pathol. Int.
- HDAC1 was found to enhance the resistance of cancer cells to oxidative stress by negatively regulating the expression of Thioredoxin Binding Protein-2 (TBP-2). It was also found that HDAC1 is located on the TBP-2 gene promoter by a protein complex composed of RFP and transcription factor NF-Y.
- RFP is useful as a biomarker (index) for estimating the prognosis of cancer patients.
- index for estimating the prognosis of cancer patients.
- endometrial cancer it was suggested that the expression of RFP is a useful indicator for determining the surgical procedure.
- An action enhancer comprising a compound that suppresses the expression of RFP (RET finger protein) gene or the action of RFP as an active ingredient, which is an action enhancer of an anticancer agent having an ability to induce oxidative stress.
- the compound is a compound selected from the group consisting of the following (a) to (d): (a) siRNA targeting the RFP gene; (b) a nucleic acid construct that generates siRNA targeting the RFP gene in the cell; (c) an antisense nucleic acid that targets a transcript of the RFP gene; (d) Ribozymes that target RFP gene transcripts.
- a method for enhancing the action of an anticancer agent having an ability to induce oxidative stress comprising a step of suppressing the expression of RFP gene or the action of RFP in a target cancer cell.
- a cancer treatment method comprising the steps of administering the action enhancer according to [1] or [2] and administering an anticancer agent having an ability to induce oxidative stress.
- a biomarker for estimating the prognosis of cancer patients comprising RFP.
- the action enhancer of the present invention is used in combination, the action of anticancer agents having oxidative stress inducing ability, such as cisplatin, can be enhanced. Thereby, the usage-amount of an anticancer agent and the kind of anticancer agent (when using two or more types of anticancer agents together) can be reduced. In addition, treatment results can be improved.
- HDAC1 and RFP Functional interaction between HDAC1 and RFP.
- HDAC1 knockdown cells have a greater decrease in cell survival index due to treatment than controls.
- endogenous RFP knockdown by siRNA Protein samples obtained from HeLa cells into which control or RFP siRNA was introduced were analyzed by Western blot using an anti-RFP antibody. Anti- ⁇ -actin antibody was used as a protein amount control. Inhibition of RFP expression was observed in cells into which siRFP had been introduced.
- HDAC1 interacts with RFP.
- HEK293 cells were transiently co-transfected with HDAC1-V5 and Flag-RFP.
- Cell lysates were immunoprecipitated with anti-V5 antibody (upper panel) or anti-Flag antibody and immunoprecipitates were analyzed by Western blot. The anti-HA antibody was used as a mock.
- HDAC1 and RFP were immunoprecipitated with anti-HDAC1 (upper panel) and anti-RFP antibody (lower panel), respectively. The immunoprecipitate was analyzed by Western blot using anti-HDAC1 antibody and anti-RFP antibody.
- RFP domain required for interaction between HDAC1 and RFP Left panel: Schematic diagram of RFP. Ring-B-box domain, coiled-coil domain and Rfp domain are shown. A Flag tag sequence was added to the cDNA corresponding to each domain.
- HDAC1-V5 and Flag-tagged RFP deletion constructs were transiently co-transfected with HDAC1-V5 and Flag-tagged RFP deletion constructs, then immunoprecipitated with anti-V5 antibody and immunoblotted with anti-Flag antibody or anti-V5 antibody.
- the pFlag vector was used as a control.
- HDAC1 region required for interaction between HDAC1 and RFP Left panel: Schematic diagram of HDAC1. The cDNA fragment corresponding to each region of HDAC1 was labeled with a V5 tag.
- HEK293 cells were transiently co-transfected with Flag-RFP and V5-tagged HDAC1 deletion constructs, immunoprecipitated with anti-Flag antibody, and immunoblotted with anti-V5 antibody or anti-Flag antibody. Arrowheads indicate co-precipitation fragments.
- a pcDNA vector was used as a control.
- TBP-2 is a common target gene for HDAC1 and RFP and causes increased sensitivity of cancer cells to H2O2 and cisplatin.
- (a) TBP-2 expression is increased by knockdown of HDAC1 and RFP.
- RNA or protein samples obtained from HeLa cells transfected with control, HDAC1 or RFP siRNA were analyzed by RT-PCR (a) or Western blot (b).
- TBP-2 was increased by knockdown of HDAC1 or RFP in both RT-PCR and Western blot.
- (d) The effect of overexpression of TBP-2 on the sensitivity of cancer cells to H 2 O 2 and cisplatin. An expression vector of TBP-2 to which a V5 tag was added was introduced into HeLa cells and treated with H 2 O 2 (10 hours) (d) and cisplatin (24 hours) (e) at the concentrations described. Whether the cells were alive or not was determined by the WST-1 method.
- HDAC1 and RFP regulate the expression of TBP-2 in colorectal and breast cancer cell lines and affect the sensitivity of cells to H 2 O 2 or cisplatin.
- Control, HDAC1, and RFP siRNA were introduced into colorectal cancer cell lines (SW480, HT-29) and breast cancer cell line (MDA-MB-231), and the proteins obtained from the cells were analyzed by Western blot. . In each cell line, knockdown of HDAC1 or RFP was found to increase TBP-2 expression.
- HDAC1 and RFP are located in the TBP-2 promoter. Each antibody described was used for immunoprecipitation.
- PCR was used to detect the TBP-2 promoter region.
- “Input” in the figure represents the PCR amplification product of the total input DNA.
- HDAC1 that deacetylates histones H3 and H4 is placed in the TBP-2 promoter by RFP.
- Cross-linked chromatin prepared from shNC1 cells and shRFP30 cells was immunoprecipitated. PCR was performed by the method described in a.
- RFP interacts with NF-YC.
- HEK293 cells were transiently co-transfected with Flag-RFP and NF-YC-V5.
- Cell lysates were immunoprecipitated with anti-V5 antibody (top) or anti-Flag antibody (bottom) and immunoprecipitates were analyzed by Western blot.
- the arrow indicates V5-NF-YC.
- the arrowhead indicates Flag-RFP.
- the anti-HA antibody was used as a mock.
- Spider endogenous RFP interacts with NF-YC. Immunoprecipitates with anti-RFP antibody or control IgG were analyzed by Western blot using anti-RFP antibody and anti-NF-YC antibody.
- HEK293 cells were co-transfected with Flag-HDAC1, Flag-RFP and NF-YC-V5 and then immunoprecipitated with anti-V5 antibody.
- the immunoprecipitate was subjected to Western blot analysis.
- the anti-HA antibody was used as a mock.
- NF-Y binds to the region near the gene of TBP-2 promoter. Immunoprecipitation analysis was performed using anti-NF-YB antibody. PCR was performed by the method described in a.
- NF-YC places HDAC1 and RFP in the TBP-2 promoter.
- Cross-linked chromatin prepared from cells transfected with control or NF-YC siRNA was immunoprecipitated with anti-HDAC1 antibody or anti-RFP antibody.
- PCR was performed by the method described in a.
- a HDAC1 / RFP / NF-YC complex formed on the TBP-2 promoter is schematically shown. RFP mediates the formation of the HDAC1 / RFP / NF-YC complex.
- HDAC1, RFP and NF-YC were co-fractionated on a gel filtration column.
- the HeLa cell extract was separated by Superose® 6 gel filtration chromatography, and each fraction was subjected to SDS-PAGE. Subsequently, immunoblotting was performed using anti-HDAC1 antibody, anti-RFP antibody, and anti-NF-YC antibody. The elution position of the molecular weight marker is shown above.
- RFP expression promotes complex formation.
- HEK293 cells were co-transfected with NF-YC-V5, HDAC1-myc and Flag-RFP and then immunoprecipitated with anti-V5 antibody. Immunoprecipitates were analyzed by Western blot.
- NF-YC-V5 was co-transfected into HeLa cells with control or RFP siRNA. Cell lysates were immunoprecipitated with anti-V5 antibody and then analyzed by Western blot. The anti-HA antibody was used as a mock. Multimerization of RFP.
- RFP forms a homo-oligomer. HEK293 cells were co-transfected with GFP-RFP and Flag-RFP and then immunoprecipitated with anti-Flag antibody. Subsequently, immunoblotting was performed using anti-Flag antibody and anti-GFP antibody. The anti-HA antibody was used as a mock. WCL represents whole cell lysate.
- Ring-B-box and coiled-coil are necessary for multimerization.
- HEK293 cells were co-transfected with an RFP deletion construct with GFP-RFP and Flag tag and then immunoprecipitated with anti-Flag antibody.
- the pFag vector was used as a control.
- Arrowheads indicate RFP constructs with a Flag tag.
- RFP is a novel target for cancer treatment.
- (a), (b), (c) RFP knockdown enhances tumor anticancer drug sensitivity in vivo.
- Three cell lines of shNC1 (control), shRFP23, and shRFP26 were transplanted subcutaneously into nude mice. When the tumor volume reached 50 mm 3 , the mice were randomly divided into a cisplatin treated group and an untreated group.
- mice were dosed intraperitoneally every 4 days with cisplatin (treatment group) or vehicle (saline: no treatment group) at a dose of 1 mg / kg.
- treatment group cisplatin
- vehicle saline: no treatment group
- RFP expression correlates with poor prognosis in colorectal cancer patients.
- the overall survival of 115 colorectal cancer patients was stratified by the presence or absence of RFP expression and analyzed by the Kaplan-Meier method. The log-rank test showed that RFP expression was significantly correlated with poor prognosis.
- A Comparison across patient groups.
- B Comparison in stage I patient group.
- A Comparison across patient groups.
- the first aspect of the present invention relates to an action enhancer for anticancer agents (hereinafter referred to as “the drug of the present invention”).
- the agent of the present invention enhances the action of an anticancer agent having an ability to induce oxidative stress.
- the agent of the present invention enhances the action of an anticancer agent whose efficacy becomes weaker or ineffective when the resistance to oxidative stress is increased in the target cancer cells.
- “enhancing the action of an anticancer agent” or “enhancing the action of an anticancer agent” means enhancing the anticancer action of the anticancer agent.
- “Improving therapeutic results” here includes (1) increasing therapeutic effects, (2) improving response rates or effective rates, and (3) reducing or avoiding side effects (according to the present invention, At least one of which is achieved).
- anticancer agents are platinum preparations such as cisplatin, carboplatin, oxaliplatin, cyclophosphamide, fluorouracil (5-FU), etoposide, doxorubicin, bleomycin, mitomycin.
- HDAC1 enhances resistance to oxidative stress of cancer cells by negatively controlling the expression of Thioredoxin binding protein 2 (TBP-2).
- TBP-2 Thioredoxin binding protein 2
- the agent of the present invention targets RFP that has been found to be necessary for the placement of HDAC1 on the TBP-2 gene promoter.
- the drug of the present invention contains a compound that suppresses the expression of RFP gene or the action of RFP as an active ingredient.
- the two terms “suppression” and “inhibition” have overlapping meanings and are often used interchangeably. Therefore, in this specification, the term “suppression” is used in a unified manner unless it is particularly necessary to distinguish between contexts.
- RFP is a nuclear protein with a ring finger structure that is activated as a cancer protein by forming a fusion protein with RET (Takahashi, M. and Cooper, G. M .: ret transforming gene encodes a fusion protein homologous to tyrosine aseskinases. Mol. Cell. Biol. 7, 1378-1385 1987 (1987) (Non-patent document 18); RFP has strong transcriptional repression activity and interacts with various nuclear proteins (Shimono, Y., Murakami, H., Hasegawa, Y. and Takahashi, M .: RFP is a transcriptional repressor and interacts with enhancer of polycomb that has dual transcriptional functions. J. Biol Chem.
- Non-Patent Document 19 Shimono, Y., Murakami, H., Kawai, K., Wade, PA, Shimokata, K . And Takahashi, M .: Mi-2 associates with BRG1 and RET finger protein at the distinct regions with transcriptional activating and repressing abilities. J. Biol. Chem.
- Non-Patent Document 20 K., Shimono, Y., Shimokata, K., Ishiguro, N., and Takahashi, M .: Microspherule protein 1, Mi-2 ⁇ , and RET finger protein associate in the nucleolus and up-regulate ribosomal gene transcription Biol. Chem. 280: 39394-39447 (2005) (Non-patent Document 21)). It has been reported that RFP is highly expressed in cancer cell lines. However, the expression state of RFP in cancer tissues has not been clarified.
- RFP gene SEQ ID NO: 1
- sequence of the mRNA of RFP SEQ ID NO: 2
- amino acid sequence of RFP SEQ ID NO: 3
- knockout mice targeting RFP do not show a serious phenotype
- targeting RFP is also preferable from the viewpoint of safety.
- high expression is observed in multiple types of human tumors or most of the investigated tumor-derived cultured cell lines (unpublished data and Non-Patent Document 16), it is expected to exert effects on a wide range of tumors. it can.
- the compound that suppresses the expression of the RFP gene which is an active ingredient of the present invention, is a compound that suppresses the expression process of the RFP gene (including transcription, post-transcriptional regulation, translation, and post-translational regulation). Examples of the compound are as follows.
- “suppression of expression” may be either transient suppression or permanent suppression.
- siRNA targeting RFP gene (b) Nucleic acid constructs that generate siRNA targeting the RFP gene in cells (c) Antisense nucleic acids that target RFP gene transcripts (d) Ribozymes that target RFP gene transcripts
- RNAi RNA interference
- the agent of the present invention containing the compound (a) or (b) as an active ingredient, the expression of RFP can be suppressed by RNAi.
- RNAi is a sequence-specific post-transcriptional gene repression process that can occur in eukaryotic cells.
- siRNA short double-stranded RNA
- siRNAs are 21-23 base pairs.
- dsRNA double-stranded RNA
- sequence-specific pathways relatively long dsRNAs are split into short interfering RNAs (ie siRNAs).
- ie siRNAs short interfering RNAs
- a sequence non-specific pathway is considered to be caused by an arbitrary dsRNA regardless of the sequence as long as it has a predetermined length or more.
- dsRNA is activated by two enzymes, namely PKR, which stops all protein synthesis by phosphorylating the translation initiation factor eIF2, and 2 ', 5' oligoadenyl, which is involved in the synthesis of RNAase L activation molecules.
- RNA double stranded RNA
- RNAi RNA composed of a sense RNA homologous to a part of the mRNA sequence of the target gene and an antisense RNA complementary thereto is introduced into the cell or expressed in the cell.
- a siRNA composed of a sense RNA homologous to a part of the mRNA sequence of the target gene and an antisense RNA complementary thereto is introduced into the cell or expressed in the cell.
- the above (a) is a compound corresponding to the former method
- the above (b) is a compound corresponding to the latter method.
- the siRNA targeting the target gene usually hybridizes with a sense RNA consisting of a sequence homologous to a continuous region in the mRNA sequence of the gene and an antisense RNA consisting of its complementary sequence. Double stranded RNA.
- the length of the “continuous region” here is usually 15 to 30 bases, preferably 18 to 23 bases, more preferably 19 to 21 bases.
- the length of the base forming the overhang is not particularly limited, but is preferably 2 bases (for example, TT, UU).
- SiRNA consisting of modified RNA may be used.
- modifications herein include phosphorothioation and the use of modified bases (eg, fluorescently labeled bases).
- SiRNA can be designed and prepared by conventional methods. In designing siRNA, a sequence unique to a target sequence (continuous sequence) is usually used. Note that programs and algorithms for selecting an appropriate target sequence have been developed.
- siRNA sequences targeting the RFP gene are listed below. 5′-GAGTTACTCGGGAGGGAAA-3 ′ (SEQ ID NO: 4. Sequence used in Examples described later) 5'-AACTCTTAGGCCTAACCCAGA-3 '(SEQ ID NO: 5. Krutzfeldt M, Ellis M, Weekes DB, Bull JJ, Eilers M, Vivanco MD, Sellers WR, Mittnacht S. (2005). Selective ablation of retinoblastoma protein function by the RET finger protein. Mol Cell.
- nucleic acid construct that generates siRNA in a cell refers to a nucleic acid molecule that, when introduced into a cell, produces a desired siRNA (siRNA that causes RNAi against a target gene) by a process in the cell.
- siRNA siRNA that causes RNAi against a target gene
- shRNA short hairpin RNA
- shRNA has a structure (hairpin structure) in which a sense RNA and an antisense RNA are linked via a loop structure part, and the loop structure part is cleaved in a cell to form a double-stranded siRNA, resulting in an RNAi effect.
- the length of the loop structure is not particularly limited, but is usually 3 to 23 bases.
- nucleic acid construct is a vector that can express a desired siRNA.
- Such vectors include those that express shRNA that is converted to siRNA by a later process (inserted with a sequence encoding shRNA) (referred to as stem loop type or short hairpin type), sense RNA and antisense RNA.
- stem loop type or short hairpin type a sequence encoding shRNA
- sense RNA a sequence encoding shRNA
- antisense RNA are vectors that are expressed separately (referred to as tandem type).
- tandem type Those skilled in the art can prepare these vectors according to conventional methods (Brummelkamp TR et al. (2002) Science 296: 550-553; Lee NS et al. (2001) Nature Biotechnology 19: 500-505; Miyagishi M & Taira K (2002) Nature Biotechnology 19: 497-500; Paddison PJ et al. (2002) Proc. Natl.
- RNAi vectors are available. You may decide to construct
- a desired RNA for example, shRNA
- SEQ ID NO: 110 A specific example of shRNA is shown in SEQ ID NO: 110 in the Sequence Listing.
- SEQ ID NO: 10 is the sequence of a single-stranded oligonucleotide (sense strand) encoding shRNA targeting RFP.
- the origin and structure of the vector are not limited as long as it has a function of generating siRNA that exerts RNAi action on the target gene in the cell. Accordingly, various viral vectors (adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, Sendai virus vectors, etc.), non-viral vectors (liposomes, positively charged liposomes, etc.) and the like can be used. it can.
- promoters that can be used in the vector are U6 promoter, H1 promoter, and tRNA promoter. These promoters are RNA polymerase III type promoters, and high expression efficiency can be expected.
- (C) is a compound used for expression suppression by the antisense method.
- the agent of the present invention containing the compound (c) as an active ingredient, the expression of RFP can be suppressed by an antisense method.
- an antisense method for example, an antisense construct that generates RNA complementary to a unique part of mRNA encoding RFP when transcribed in a target cell is used. Such an antisense construct is introduced into a target cell, for example, in the form of an expression plasmid.
- an oligonucleotide probe that hybridizes with an mRNA / or genomic DNA sequence encoding RFP and inhibits its expression when introduced into a target cell can also be employed.
- an oligonucleotide probe one that is resistant to endogenous nucleases such as exonuclease and / or endonuclease is preferably used.
- oligodeoxyribonucleotide derived from a region containing a translation start site for example, a region of ⁇ 10 to +10 of mRNA encoding RFP is preferable.
- the complementarity between the antisense nucleic acid and the target nucleic acid is exact, but some mismatch may exist.
- the ability of an antisense nucleic acid to hybridize to a target nucleic acid generally depends on both the degree of complementarity and the length of both nucleic acids. Usually, the longer the antisense nucleic acid used, the more stable duplexes (or triplexes) can be formed with the target nucleic acid, even if the number of mismatches is large.
- One skilled in the art can ascertain the degree of acceptable mismatch using standard techniques.
- the antisense nucleic acid may be DNA, RNA, or a chimeric mixture thereof, or a derivative or modified form thereof. Moreover, it may be single-stranded or double-stranded. By modifying the base moiety, sugar moiety, or phosphate skeleton moiety, the stability, hybridization ability, etc. of the antisense nucleic acid can be improved.
- antisense nucleic acids can be used to promote cell membrane transport (eg, Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No.
- the antisense nucleic acid can be synthesized by a conventional method, for example, using a commercially available automatic DNA synthesizer (for example, Applied Biosystems). For example, Stein et al. (1988), Nucl. Acids Res. 16: 3209 and Sarin et al., (1988), Proc. Natl. Acad. Sci. USA 85: 7448- 7451 etc. can be referred to.
- Strong promoters such as pol II and pol III can be used to enhance the action of antisense nucleic acids in target cells. That is, when a construct containing an antisense nucleic acid arranged under the control of such a promoter is introduced into a target cell, a sufficient amount of the antisense nucleic acid can be transcribed by the action of the promoter. Expression of the antisense nucleic acid can be performed by any promoter (inducible promoter or constitutive promoter) known to function in mammalian cells (preferably human cells).
- the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter from the 3 ′ end region of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), herpes zoster thymidine A promoter such as a kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445) can be used.
- expression suppression by a ribozyme is used (in the case of the compound (d) above).
- the target mRNA can be destroyed using a ribozyme that cleaves the mRNA with a site-specific recognition sequence, but preferably a hammerhead ribozyme is used.
- a ribozyme for example, Haseloff and Gerlach, 1988, Nature, 334: 585-591 can be referred to.
- a ribozyme may be constructed using a modified oligonucleotide.
- a nucleic acid construct in which DNA encoding the ribozyme is placed under the control of a strong promoter for example, pol II or pol III
- a strong promoter for example, pol II or pol III
- a compound that suppresses the action of RFP is used as an active ingredient.
- RFP is involved in the control of oxidative stress sensitivity by HDAC1 through physical and functional interaction with HDAC1.
- the drug of this embodiment suppresses the interaction between RFP and HDAC1, and enhances the action of the anticancer drug.
- the kind of the compound is not limited as long as the interaction between RFP and HDAC1 can be suppressed.
- the N-terminal side of HFAC1 binds to the coiled-coil domain and Rfp domain of RFP.
- a compound that can inhibit the binding may be used as an active ingredient.
- an antibody that specifically binds to the coiled-coil domain or Rfp domain of RFP can be employed.
- the type and form of the antibody are not particularly limited. Any of a polyclonal antibody, an oligoclonal antibody and a monoclonal antibody may be used. Chimeric antibodies (eg, human and mouse chimeras), humanized antibodies, human antibodies, and the like can also be used. Furthermore, antibody fragments such as Fab, Fab ′, F (ab ′) 2 , scFv and dsFv can also be used.
- the antibody as an active ingredient can be prepared by an immunological technique, a phage display method, a ribosome display method or the like using HDAC1 or RFP or a part thereof (preferably a site involved in interaction) as an antigen.
- a compound that competitively binds to RFP on the N-terminal side of HDAC1 may be used as an active ingredient.
- examples of such compounds are RFP coiled-coil domain-like polypeptides and Rfp domain-like polypeptides.
- the antibody itself may not be used as an active ingredient, but a vector capable of expressing a desired antibody in cells may be used as an active ingredient. The same applies to compounds other than antibodies.
- the preparation of the drug of the present invention can be performed according to a conventional method.
- other pharmaceutically acceptable ingredients for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like.
- excipient lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used.
- disintegrant starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer.
- emulsifier gum arabic, sodium alginate, tragacanth and the like can be used.
- suspending agent glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
- soothing agent benzyl alcohol, chlorobutanol, sorbitol and the like can be used.
- stabilizer propylene glycol, diethylin sulfite, ascorbic acid or the like can be used.
- preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used.
- preservatives benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
- the dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories.
- the drug of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (or preventive effect) (that is, a therapeutically effective amount).
- the amount of the active ingredient in the drug of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, within the range of about 0.1 wt% to about 95 wt% so as to achieve a desired dose.
- the drug of the present invention is administered to the subject by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on the dosage form.
- oral administration or parenteral administration intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.
- parenteral administration intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.
- these administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed).
- a nucleic acid construct is used as an active ingredient (for example, an embodiment using RNAi), not only in vivo administration but
- the “subject” to which the agent of the present invention is administered is not particularly limited, and includes humans and non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows) Pigs, goats, sheep, dogs, cats, chickens, quails, etc.).
- the medicament of the present invention is applied to humans.
- the dosage varies depending on the patient's symptoms, age, sex, weight, etc., those skilled in the art can appropriately set an appropriate dosage. In setting the administration schedule, it is possible to consider patient symptoms and duration of drug effect.
- the agent of the present invention is used in combination with an anticancer agent. That is, the drug of the present invention and the anticancer agent are administered in combination.
- the agent of the present invention and a predetermined anticancer agent are prepared, and both are administered to the subject. In this case, both agents are administered (applied) to the subject simultaneously or at a predetermined time interval. “Simultaneous” here does not require strict simultaneity. Therefore, when both agents are administered to the subject after mixing both agents, for example, both agents are administered under the condition that there is no difference in time. In this case, the concept of “simultaneous” is also included in the case where the present invention is carried out under a condition without a time difference.
- the anticancer drug is administered after a predetermined time after the drug of the present invention is administered.
- the predetermined time here is, for example, 1 hour to 72 hours (specific example is 48 hours).
- the administration of the drug of the present invention is preceded, the action according to the present invention is easily exerted, and a good treatment result is obtained.
- the expression of RFP gene or the action of RFP is suppressed in the target cancer cells.
- suppression of TBP-2 expression by HDAC1 is suppressed or released, and as a result, the sensitivity of the target cancer cells to oxidative stress increases.
- the state in which the anticancer agent is likely to be effective that is, the state in which the resistance to the anticancer agent is reduced, is formed, the therapeutic effect of the anticancer agent is improved.
- the drug of the present invention is expected to be applied to various cancers.
- Cancers that can be targeted by the drug of the present invention include esophageal cancer, oral cancer, maxillary cancer, laryngeal cancer, pharyngeal cancer, gastric cancer, duodenal cancer, colon cancer, hepatocellular carcinoma, cholangiocellular carcinoma, lung cancer, prostate cancer, renal cancer.
- the anticancer agent used in combination is not particularly limited as long as it has an ability to induce oxidative stress.
- anticancer agents are platinum preparations such as cisplatin, carboplatin and oxaliplatin, cyclophosphamide, fluorouracil (5-FU), etoposide, doxorubicin, bleomycin and mitomycin.
- Two or more anticancer agents can be used in combination.
- the dosage of the anticancer agent is in accordance with the amount used when it is used alone (that is, the usual amount used). However, since the enhancement of the action of the anticancer agent can be expected by the combined use with the drug of the present invention, the dose may be set lower than the normal dose.
- a person skilled in the art can set the “normal use amount” in consideration of the patient's medical condition, age, sex, weight, and the like.
- the second aspect of the present invention provides an anticancer drug resistance test method (hereinafter referred to as “test method of the present invention”) using the expression level of the RFP gene.
- test method of the present invention anticancer drug resistance test method
- the determination result is used, for example, for determination of a treatment policy (selection of an effective treatment method, etc.).
- improvement of treatment results, improvement of prognosis, improvement of patient's quality of life (QOL) and the like are brought about.
- the test method of the present invention provides extremely useful information for cancer treatment.
- the following steps are performed. That is, a step of detecting the RFP expression level in cancer cells separated from a living body (detection step), and a step of determining the resistance of the cancer cells to an anticancer agent having an oxidative stress inducing ability based on the result of the step ( Determination step).
- cancer cells separated from the living body are prepared.
- “Separated from the living body” refers to a state in which the test cancer cell is completely isolated from the living body derived from the part of the living tissue in which the test cancer cell exists. As long as this condition is satisfied, it may be a cancer cell in a state of forming a tissue with surrounding cells (that is, a state of a tissue piece) or a cancer cell separated from the surrounding cells.
- cells may be prepared in the same manner as when performing biopsy histology (biopsy), and for the latter, for example, cells may be prepared as in the case of performing cytodiagnosis.
- the expression level of RFP in the prepared cancer cells is examined.
- the expression level of RFP is usually calculated based on comparison with a control (comparison control).
- a control for control, various cancer cells whose RFP expression level is known in advance, and normal cells collected in combination with test cancer cells when collecting test cancer cells from a living body can be used. . It is not essential to strictly quantify the expression level of RFP, and it is sufficient that the expression level of RFP can be detected to the extent that the resistance of the test cancer cell to the anticancer agent can be evaluated through comparison with the control.
- the expression of RFP in the test cancer cells is expressed as RFP positive or RFP positive, and the expression of RFP in the test cancer cells is also expressed as RFP negative or RFP negative. To do.
- the boundary value (expressed by the ratio of stained cells) that separates positive and negative may be set as appropriate according to the detection method, detection conditions, and the like. For example, the boundary value can be set between 5% and 30%.
- the expression level of RFP is detected at the protein level or mRNA level.
- the former is better if high accuracy is required.
- detecting the RFP expression level at the protein level that is, when examining the RFP protein level as the RFP expression level, it is not limited to this, but Western blotting or immunohistochemistry (immunostaining) may be used.
- immunohistochemistry the morphology and distribution of test cancer cells can be examined simultaneously, and additional information can be obtained at the same time.
- an antibody that specifically recognizes RFP is used, and the amount of RFP protein is examined using the binding (binding amount) of the antibody as an index.
- Immunohistochemistry using anti-RFP antibodies is, for example, ABC (Avidin-Biotin Complex) method, PAP (Peroxydase-anti-Peroxydase Complex) method, LAB (Linked Avidin-Biotin) method, LSAB (Linked Streptavidin-Biotin) method etc. It can be carried out. Standard procols for each of these methods are well known (see, for example, “Enzyme Antibody Method, Revised 3rd Edition”, Keiichi Watanabe, Kazuho Nakane, Interdisciplinary Planning).
- the type and origin of the anti-RFP antibody used for immunohistochemistry are not particularly limited.
- a monoclonal antibody is preferably used, but an oligoclonal antibody (a mixture of several to several tens of antibodies) or a polyclonal antibody may be used as long as RFP can be detected with sufficient specificity.
- Antibody fragments such as Fab, Fab ′, F (ab ′) 2 , scFv, and dsFv antibodies may be used.
- Anti-RFP antibodies can be prepared using immunological techniques, phage display methods, ribosome display methods, and the like. It should be noted that anti-RFP is described in the document "Shimono, Y. et al.
- RET finger protein is a transcriptional repressor and interacts with enhancer of polycomb that has dual transcriptional functions.J Biol Chem. 275 (50): 39411-9 (2000)". There is a description of the preparation method of the antibody (rabbit polyclonal) and its use example, which is helpful.
- Fixation / embedding with paraffin Fix tissue collected from a living body (in the case of an autopsy case) with formalin, paraformaldehyde, or the like. Then embedded in paraffin. Generally dehydrated with alcohol, treated with xylene, and finally embedded in paraffin. The paraffin-embedded specimen is sliced to a desired thickness (eg 3-5 ⁇ m) and spread on a glass slide. A frozen specimen may be used instead of the paraffin-embedded specimen.
- Deparaffinization In general, treatment is performed sequentially with xylene, alcohol, and purified water.
- Nuclear staining Nuclear staining is performed by reacting Meyer's hematoxylin for several seconds to several tens of seconds. Wash with running water and color (usually for a few minutes). (10) Dehydration, clearing, sealing After dehydrating with alcohol, clearing with xylene, and finally sealing with synthetic resin, glycerin, rubber syrup or the like.
- RT-PCR Molecular Cloning, Third Edition, 8.46, Cold Spring Harbor Laboratory Press, New York
- Northern blot Molecular Cloning, Third Edition, 7.42
- RT-PCR Molecular Cloning, Third Edition, 8.46, Cold Spring Harbor Laboratory Press, New York
- dot blot method Molecular Cloning, Third Edition, 7.46, Cold Spring Harbor Laboratory Press, New York
- method using DNA chip DNA array
- in situ hybridization etc. it can.
- a person skilled in the art can use a nucleic acid primer or nucleic acid suitable for each method based on the RFP sequence (NCBI database, Accession: RFPNM_006510, DEFINITION: Homo sapiens tripartite motif-containing 27 (TRIM27), mRNA. (SEQ ID NO: 2)). Probes can be designed in a conventional manner.
- the resistance of the cancer cell to the anticancer agent having the ability to induce oxidative stress is determined based on the result of the detection step, that is, the RFP expression level.
- the RFP expression level the result of the detection step.
- a plurality of evaluation categories in which the RFP expression level and the anticancer drug resistance are associated are set in advance. Then, based on the RFP expression level obtained in the detection step, an evaluation category corresponding to the test cancer cell is determined.
- an example focusing on the presence or absence of RFP expression (Example 1) and an example focusing on the level of RFP expression (Example 2) are shown below.
- the number of evaluation categories, the amount of RFP expression associated with each evaluation category, and the evaluation results can be arbitrarily set through preliminary experiments and the like without being limited to the above examples. Note that the determination / evaluation in the present invention can be automatically / mechanically performed regardless of the judgment of a person having specialized knowledge such as a doctor or a laboratory technician.
- the treatment policy can be determined using the result of the determination result obtained by the test method of the present invention. For example, if the determination result is “resistant to anticancer drugs” or the determination result is “high resistance to anticancer drugs”, the anticancer drug alone has the desired therapeutic effect. It can be predicted that it cannot be demonstrated. In such a case, it is an effective therapeutic policy to administer the drug of the present invention in combination with an anticancer drug to enhance the action of the anticancer drug. On the other hand, if the determination result is “sensitive to anticancer drugs” or the determination result is “low resistance to anticancer drugs”, the therapeutic effect expected from anticancer drugs alone is exhibited. It is possible to predict that it will be possible. In such cases, administration of an anticancer agent alone is one of the treatment policy options. However, even in such a case, the action enhancer of the present invention may be administered in combination in order to further improve the therapeutic effect by enhancing the action of the anticancer agent.
- the third aspect of the present invention provides a marker for prognosis estimation of cancer patients, comprising RFP.
- the biomarker is useful for understanding the prognosis of cancer patients. It also serves as a guideline for determining the surgical technique. For example, when RFP expression is observed in a specimen (cancer cell) collected from a patient with endometrial cancer (for example, when it is judged positive by immunostaining), it is preferable to adopt a more aggressive technique. It is possible to determine. More aggressive procedures include extensive hysterectomy and lymphoid dissection.
- a prognosis estimation method for cancer patients is also provided as an application of the biomarker.
- information useful for estimating the prognosis of a cancer patient can be obtained.
- the information is used, for example, for determining a treatment policy (selecting an effective treatment method, etc.).
- improvement of treatment results, improvement of prognosis, improvement of patient's quality of life (QOL) and the like are brought about.
- biomarker for estimating prognosis of cancer patient refers to a biomolecule that serves as an index for estimating prognosis of a cancer patient.
- the type of “cancer” is not particularly limited. However, the biomarker of the present invention is particularly useful in estimating the prognosis of patients suffering from colon cancer or endometrial cancer.
- the biomarker of the present invention is used as a determination index.
- the prognosis of a cancer patient is estimated using the expression level of RFP in cancer cells separated from the cancer patient as an index. More specifically, a step (detection step) of detecting an RFP expression level in cancer cells separated from a cancer patient and a step (prognosis estimation step) of estimating a prognosis based on the detection result are performed. What is necessary is just to implement the detection step here similarly to the detection step of the said 2nd aspect. Examples of cancer patients are those suffering from colorectal cancer or endometrial cancer.
- the prognosis is estimated using the expression level of RFP in cancer cells as an index. Basically, a criterion is adopted that the prognosis is poor when the amount of RFP expression is large.
- specific examples of evaluation based on the expression level of RFP are shown.
- a plurality of evaluation categories in which the RFP expression level and the prognosis are associated are set in advance. Then, based on the RFP expression level obtained in the detection step, an evaluation category corresponding to the test cancer cell is determined.
- an example focusing on the presence or absence of RFP expression (Example 1) and an example focusing on the level of RFP expression (Example 2) are shown below.
- the number of evaluation categories, the amount of RFP expression associated with each evaluation category, and the evaluation results can be arbitrarily set through preliminary experiments and the like without being limited to the above examples. Note that the determination / evaluation in the present invention can be automatically / mechanically performed regardless of the judgment of a person having specialized knowledge such as a doctor or a laboratory technician.
- the present invention further provides a reagent for estimating the prognosis of a cancer patient and a kit for estimating the prognosis of a cancer patient.
- the reagent of the present invention comprises an anti-RFP antibody.
- an anti-RFP antibody For the type and origin of the anti-RFP antibody, the corresponding explanation in the second aspect is incorporated. If a labeled antibody is used as the anti-RFP antibody, it is possible to directly detect the amount of bound antibody using the labeled amount as an index. Therefore, a simpler inspection method can be constructed. On the other hand, in addition to the need to prepare an anti-RFP antibody to which a labeling substance is bound, there is a problem that detection sensitivity is generally lowered.
- an indirect detection method such as a method using a secondary antibody to which a labeling substance is bound or a method using a polymer to which a secondary antibody and a labeling substance are bound.
- the secondary antibody here is an antibody having a specific binding property to the anti-RFP antibody.
- an anti-RFP antibody is prepared as a rabbit antibody
- an anti-rabbit IgG antibody can be used.
- Labeled secondary antibodies that can be used against various types of antibodies such as rabbits, goats, and mice are commercially available (for example, Funakoshi Co., Ltd., Cosmo Bio Co., Ltd., etc.) and are appropriate for the reagents of the present invention. Can be appropriately selected and used.
- labeling substances include peroxidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -D-galactosidase, enzymes such as glucose oxidase and glucose-6-phosphate dehydrogenase, fluorescein isothiocyanate (FITC), Fluorescent materials such as tetramethylrhodamine isothiocyanate (TRITC) and europium, chemiluminescent materials such as luminol, isoluminol and acridinium derivatives, coenzymes such as NAD, biotin, and radioactive materials such as 131 I and 125 I.
- HRP horseradish peroxidase
- alkaline phosphatase ⁇ -D-galactosidase
- enzymes such as glucose oxidase and glucose-6-phosphate dehydrogenase
- FITC fluorescein isothiocyanate
- TRITC
- the reagent of the present invention is solid-phased according to its use.
- the insoluble support used for the solid phase is not particularly limited.
- a resin such as polystyrene resin, polycarbonate resin, silicon resin, nylon resin, or an insoluble support made of a water-insoluble substance such as glass can be used.
- the antibody can be supported on the insoluble support by physical adsorption or chemical adsorption.
- the kit of the present invention contains the reagent of the present invention as a main component.
- Other reagents buffers, blocking reagents, enzyme substrates, coloring reagents, etc.
- devices or instruments used in performing the test method may be included in the kit.
- RFP Reactive protein
- an instruction manual is attached to the kit of the present invention.
- Plasmid pcDNA3-HDAC1-Flag was provided by Dr. Kawaguchi (Aichi Prefecture Psychosomatic Colony). Full-length NF-YA, NF-YB, NF-YC, HDAC1, HDAC1 deletion fragment, and TBP-2 cDNA were cloned into pcDNA3.1 / V5-His-TOPO vector. The operation followed the instruction manual. HDAC1 cDNA excised from pcDNA3.1 / V5-His-HDAC1 was inserted into pcDNA3.1 / myc-His vector. The pFlag-RFP construct is as described (Reference 20). RFP cDNA was inserted into the pEGFP-C1 vector.
- RNAi HDAC1 siRNA was purchased from Dhamacon, and siRFP, siTBP-2, and siNF-YC were purchased from QIAGEN. siRNA was introduced into cells using Lipofectamine 2000 (Invitrogen). A single-stranded oligonucleotide encoding an shRNA targeting RFP and its complementary sequence were synthesized for knockdown using shRNA (the sense strand is shown below).
- shRFP 5'-GATCGAGTTACTCGGGAGGGAAATTCAAGAGATTTCCCTCCCGAGTAACTCTTTTTTGGAAA-3 '(SEQ ID NO: 10) This complementary oligonucleotide pair was inserted into pSilencer3.1-H1 neo vector (Ambion) and used as an shRNA expression vector.
- HDAC1-specific primers sense: 5′-CTCCTGTTTTTTTCAGGCTCC-3 ′ (SEQ ID NO: 11), antisense: 5′-AGGAGAAGACAGACAGAGGGC-3 ′ (SEQ ID NO: 12)
- RFP-specific primer sense: 5 '-TGCTCGACTGCGGCCATAAC-3' (SEQ ID NO: 13), antisense: 5'-TCGGTGCGCAGCTGCTTTAC-3 '(SEQ ID NO: 14)
- TBP-2 specific primer sense: 5'- TGAGATGGTGATCATGAGACC -3' (SEQ ID NO: 15)
- GAPDH specific primer sense: 5′-GTATTGACATCCACCAGATCC-3 '(SEQ
- Chromatin immunoprecipitation (ChIP) assay A chromatin immunoprecipitation assay was performed according to the method described above (Reference 28). Briefly, formaldehyde cross-linked chromatin was sonicated and the resulting chromatin fragments were immunoprecipitated with anti-HDAC1 antibody, anti-acetylated H3 antibody, anti-acetylated H4 antibody, anti-RFP antibody or anti-NFYB antibody. Purified normal rabbit or mouse IgG was used as a control. 5M NaCl was added to a final concentration of 0.2M, and then decrosslinked by incubation at 65 ° C for 8 hours. Purified DNA and total cellular DNA were amplified by PCR (32-37 cycles).
- the sample was reacted with an anti-RFP rabbit polyclonal antibody followed by a biotinylated goat secondary antibody. Further, streptavidin added with peroxidase was reacted, and color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB). A specimen in which brown staining was observed in 10% or more of the cells was determined to be RFP positive.
- the statistical analysis software Stat View was used to examine the relationship between the expression of RFP and the prognosis of colorectal cancer patients by the Kaplan-Meier method.
- HDACi affects the sensitivity of HeLa cells to oxidative stress. It was found that the sensitivity to H 2 O 2, which is an inducer of oxidative stress, was enhanced in HeLa cells treated with Trichostatin A (TSA), which is a representative HDACi (FIG. 1a). In order to elucidate the molecular mechanism involved in the enhancement of sensitivity by HDACi, research was conducted focusing on HDAC1. When HDAC1 was knocked down with siRNA, the cytotoxic effects of H 2 O 2 and cisplatin were significantly enhanced (FIGS. 1b-d). This suggests that HDACi may increase oxidative stress sensitivity through inhibition of HDAC1 function.
- TSA Trichostatin A
- HDAC1 The molecular mechanism of stress sensitivity control by HDAC1 is presumed to be through the interaction with p53 (References 16-19), but in the HeLa cells used this time, p53 expression is caused by the E6 protein of papillomavirus. Since it is suppressed, there is a high possibility that it is controlled via another mechanism.
- RFP which is highly expressed in tumor cell lines, interacts with HDAC1 and shows co-localization within the cell (Reference 20). This suggests that RFP may be involved in the regulation of oxidative stress sensitivity by HDAC1.
- HDAC1 When the interaction domain of RFP and HDAC1 was first examined, it was found that the N-terminal side of HDAC1 binds to the coiled-coil domain and Rfp domain of RFP (FIG. 2).
- knockdown of RFP increases the sensitivity of HeLa cells to H 2 O 2 and cisplatin as in HDAC1 (FIG. 1b-g). This suggested that RFP and HDAC1 interacted physically and functionally.
- TBP-2 binds to Thioredoxin having the function of removing ROS and inhibits the function (Reference Documents 22 and 23), and enhances sensitivity to oxidative stress and cisplatin by overexpression (Reference Document 4). 24) is known. From the results of RT-PCR and Western blot, it was confirmed that the expression of TBP-2 was regulated at the transcription level by HDAC1 and RFP (FIGS. 3a and b). Here, it was examined whether the expression of TBP-2 actually affects the sensitivity to H 2 O 2 and cisplatin in HeLa cells. When TBP-2 was overexpressed in HeLa cells, a marked increase in sensitivity was observed (FIGS. 3d and e).
- HDAC1 and RFP directly regulate the expression of TBP-2.
- HDAC1 and RFP were arranged in the gene vicinity region of the TBP-2 promoter.
- HDACi transcriptional repression by RFP is inhibited by HDACi (results not shown).
- the expression of TBP-2 was increased by RFP knockdown, suggesting that histone acetylation in the TBP-2 promoter region was enhanced.
- HDAC1 and RFP are not located in the TBP-2 promoter through interaction with DNA-binding proteins such as transcription factors, as there is no report that binds directly to DNA for HDAC1 and RFP. I thought.
- the TBP-2 promoter contains the binding sequence of transcription factor NF-Y (Nuclear Factor Y) in its sequence, and it is reported that NF-Y is required when TBP-2 expression is induced by HDACi (Reference 15). From this, it was speculated that NF-Y might be required when HDAC1 and RFP are placed in the TBP-2 promoter.
- NF-Y is known to be a trimeric transcription factor composed of three proteins: NF-YA, NF-YB, and NF-YC.
- RFP may be complexed by mediating the interaction between HDAC1-NF-YC.
- 6b and 6c show that the interaction between HDAC1-NF-YC is enhanced in cells exogenously expressing RFP, and attenuated in cells knocking down RFP. It was. Furthermore, the state of RFP in this complex was examined. RFP has been reported to multimerize (Refs. 25 and 26). From the results of this experiment, Ring-B-box domain (RB) and Coiled-coil domain (CC) ) was necessary (FIG. 7b).
- HDAC1 regulates apoptosis induced by various stresses through interaction with p53 (refs. 16-19).
- HDAC1 regulates the oxidative stress sensitivity of cancer cells by forming a protein complex with RFP and NF-Y and suppressing the expression of TBP-2.
- the increased expression of TBP-2 attenuates the ROS removal effect of Thioredoxin, making cancer cells sensitive to oxidative stress induced by anticancer drugs. From these results, it is considered that HDAC1 imparts anticancer drug resistance to cancer cells by controlling the antioxidant mechanism.
- the action enhancer of the present invention provides a cancer treatment method with few side effects.
- the action enhancer of the present invention is used in combination with an anticancer agent having an ability to induce oxidative stress.
- the action enhancer of the present invention can also be applied when two or more kinds of anticancer agents are used in combination.
- Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function.J Immunol. 164 (12): 6287-95 (2000). 5. Xu, WS., Purgiani, RB. And Marks, PA.Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene. 26 (37): 5541-52 (2007). 6.Marks, PA. And Breslow, R. Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug.Nature Biotechnology. 25 (1): 84-90 (2007). 7. Miyajima, A. et al.
- 1,25-Dihydroxyvitamin D3 enhances the susceptibility of breast cancer cells to doxorubicin-induced oxidative damage.Cancer Res. 59 (4): 862-7 (1999). 11.Godwin, AK. Et al. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis.Proc Natl Acad Sci U S A. 89 (7): 3070-4 (1992). 12. Yokomizo, A. et al. Cellular levels of thioredoxin associated with drug sensitivity to cisplatin, mitomycin C, doxorubicin and etoposide.Cancer Res. 55 (19): 4293-6 (1995). 13. Sasada, T. et al.
- Mi-2 beta associates with BRG1 and RET finger protein at the distinct regions with transcriptional activating and repressing abilities.
- J Biol Chem. 278 (51): 51638-45 (2003).
- 20. Shimono, Y. et al. RET finger protein is a transcriptional repressor and interacts with enhancer of polycomb that has dual transcriptional functions.
- RT.Vitamin D (3) -up-regulated protein-1 is a stress-responsive gene that regulates cardiomyocyte viability through interaction with thioredoxin.J Biol Chem. 277 ( 29): 26496-500 (2002). 23. Baker, AF. Et al. Identification of thioredoxin-interacting protein 1 as a hypoxia-inducible factor 1 alpha-induced gene in pancreatic cancer. Pancreas. 36 (2): 178-86. (2008). 24. Cao, T., Borden, KL., Freemont, PS.
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Abstract
Description
[1]酸化ストレス誘導能を有する抗がん剤の作用増強剤であって、RFP(RET finger protein)遺伝子の発現又はRFPの作用を抑制する化合物を有効成分とする作用増強剤。
[2]前記化合物が以下の(a)~(d)からなる群より選択される化合物である、[1]に記載の作用増強剤:
(a)RFP遺伝子を標的とするsiRNA;
(b)RFP遺伝子を標的とするsiRNAを細胞内で生成する核酸コンストラクト;
(c)RFP遺伝子の転写産物を標的とするアンチセンス核酸;
(d)RFP遺伝子の転写産物を標的とするリボザイム。
[3]酸化ストレス誘導能を有する抗がん剤と併用される、[1]又は[2]に記載の作用増強剤。
[4]酸化ストレス誘導能を有する抗がん剤の作用を増強する方法であって、標的がん細胞内において、RFP遺伝子の発現又はRFPの作用を抑制するステップを含む方法。
[5][1]又は[2]に記載の作用増強剤を投与するステップと、酸化ストレス誘導能を有する抗がん剤を投与するステップと、を含むがん治療法。
[6]生体から分離されたがん細胞内のRFP発現量を調べるステップと、
前記ステップの結果に基づき、酸化ストレス誘導能を有する抗癌剤に対する前記がん細胞の耐性を判定するステップと、を含む抗がん剤耐性検査法。
[7]RFPからなる、がん患者の予後推定用バイオマーカー。
[8]がんが大腸癌又は子宮体がんである、[7]に記載の予後推定用バイオマーカー。
[9]がん患者から分離されたがん細胞内のRFP発現量を指標とした、がん患者の予後推定法。
[10]がん細胞内のRFP発現量が多いことが、予後が不良であることを示す、[9]に記載の予後推定法。
[11]がんが大腸癌又は子宮体がんである、[9]又は[10]に記載の予後推定法。
[12]抗RFP抗体からなる、がん患者の予後推定用試薬。
[13]がんが大腸癌又は子宮体がんである、[12]に記載の予後推定用試薬。
[14][12]又[13]に記載の試薬を含む、がん患者の予後推定用キット。
[15]がんが大腸癌又は子宮体がんである、[14]に記載の予後推定用キット。
本発明の第1の局面は抗がん剤の作用増強剤(以下、「本発明の薬剤」と称する)に関する。本発明の薬剤は、酸化ストレス誘導能を有する抗がん剤の作用を増強する。別の言い方をすれば、本発明の薬剤は、標的のがん細胞において酸化ストレス抵抗性が亢進すると効き目が弱くなる又は効かなくなる抗がん剤の作用を増強する。本発明において「抗がん剤の作用を増強する」又は「抗がん剤の作用増強」とは、抗がん剤の抗がん作用を高めることをいう。本発明の薬剤を抗がん剤と併用することによって、がんの治療成績が向上する。ここでの「治療成績の向上」には、(1)治療効果の増大、(2)奏功率ないし有効率の向上、(3)副作用の低減ないし回避が含まれる(本発明によれば、この中の少なくとも一つが達成される)。抗がん剤の例は、シスプラチン、カルボプラチン、オキサリプラチン等のプラチナ製剤、サイクロフォスファミド、フルオロウラシル(5-FU)、エトポシド、ドキソルビシン、ブレオマイシン、マイトマイシンである。
(a)RFP遺伝子を標的とするsiRNA
(b)RFP遺伝子を標的とするsiRNAを細胞内で生成する核酸コンストラクト
(c)RFP遺伝子の転写産物を標的とするアンチセンス核酸
(d)RFP遺伝子の転写産物を標的とするリボザイム
5'-GAGTTACTCGGGAGGGAAA-3'(配列番号4。後述の実施例で使用した配列)
5'-AACTCTTAGGCCTAACCCAGA-3'(配列番号5。Krutzfeldt M, Ellis M, Weekes DB, Bull JJ, Eilers M, Vivanco MD, Sellers WR, Mittnacht S. (2005). Selective ablation of retinoblastoma protein function by the RET finger protein. Mol Cell. 18(2):213-24.を参照)
5'-AAGAGAGGCUCAGUUAUACUC-3'(配列番号6。Krutzfeldt M, Ellis M, Weekes DB, Bull JJ, Eilers M, Vivanco MD, Sellers WR, Mittnacht S. (2005). Selective ablation of retinoblastoma protein function by the RET finger protein. Mol Cell. 18(2):213-24.を参照)
5'-CCCUAUGAGUGGGAUUGAU-3'(配列番号7。Fukushige S, Kondo E, Gu Z, Suzuki H, Horii A.(2006).RET finger protein enhances MBD2- and MBD4-dependent transcriptional repression. Biochem Biophys Res Commun. 351(1):85-92. Epub 2006 Oct 10.を参照)
5'-GACTCAGTGTGCAGAAAAG-3'(配列番号8。Zha J, Han KJ, Xu LG, He W, Zhou Q, Chen D, Zhai Z, Shu HB.(2006). The Ret finger protein inhibits signaling mediated by the noncanonical and canonical IkappaB kinase family members. J Immunol. Jan 15;176(2):1072-80.を参照)
5'-AGAACCAGCTCGACCATT-3'(配列番号9。Townson SM, Kang K, Lee AV, Oesterreich S.(2006). Novel role of the RET finger protein in estrogen receptor-mediated transcription in MCF-7 cells. Biochem Biophys Res Commun. Oct 20;349(2):540-8. Epub 2006 Aug 22.を参照)
アンチセンス核酸は例えば市販の自動DNA合成装置(例えばアプライド・バイオシステムズ社等)を使用するなど、常法で合成することができる。核酸修飾体や誘導体の作製には例えば、Stein et al.(1988), Nucl. Acids Res. 16:3209やSarin et al., (1988), Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451等を参照することができる。
アンチセンス核酸の発現は、哺乳動物細胞(好ましくはヒト細胞)で機能することが知られている任意のプロモーター(誘導性プロモーター又は構成的プロモーター)によって行うことができる。例えば、SV40初期プロモーター領域 (Bernoist and Chambon, 1981, Nature 290:304-310)、ラウス肉腫ウィルスの3'末端領域由来のプロモーター(Yamamoto et al., 1980, Cell 22:787-797)、疱疹チミジン・キナーゼ・プロモーター(Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445)等のプロモーターを使用することができる。
アンチセンス法を利用する場合と同様に、例えば安定性やターゲット能を向上させることを目的として、修飾されたオリゴヌクレオチドを用いてリボザイムを構築してもよい。効果的な量のリボザイムを標的細胞内で生成させるために、例えば、強力なプロモーター(例えばpol IIやpol III)の制御下に、当該リボザイムをコードするDNAを配置した核酸コンストラクトを使用することが好ましい。
有効成分としての抗体は、HDAC1若しくはRFP又はこれらの一部(好ましくは相互作用に関与する部位)を抗原として利用した免疫学的手法、ファージディスプレイ法、リボソームディスプレイ法などによって調製することができる。
尚、抗体自体を有効成分とするのではなく、細胞内で所望の抗体を発現可能なベクターを有効成分としてもよい。抗体以外の化合物についても同様である。
本発明の薬剤には、期待される治療効果(又は予防効果)を得るために必要な量(即ち治療上有効量)の有効成分が含有される。本発明の薬剤中の有効成分量は一般に剤型によって異なるが、所望の投与量を達成できるように有効成分量を例えば約0.1重量%~約95重量%の範囲内で設定する。
本発明の薬剤はその剤型に応じて経口投与又は非経口投与(静脈内、動脈内、皮下、皮内、筋肉内、又は腹腔内注射、経皮、経鼻、経粘膜など)によって対象に適用される。これらの投与経路は互いに排他的なものではなく、任意に選択される二つ以上を併用することもできる(例えば、経口投与と同時に又は所定時間経過後に静脈注射等を行う等)。
核酸コンストラクトを有効成分とした場合(例えばRNAiを利用する態様)、in vivo投与に限らず、ex vivo投与を採用することもできる。
投与量は患者の症状、年齢、性別、及び体重などによって異なるが、当業者であれば適宜適当な投与量を設定することが可能である。投与スケジュールの設定においては、患者の症状や薬剤の効果持続時間などを考慮することができる。
尚、本発明の薬剤と抗がん剤を混合した配合剤を用意し、これを対象に投与することにしてもよい。
本発明者らの検討の結果、RFPの高発現が、ヒトの大腸癌におけるTBP-2の発現低下や、大腸癌患者の予後不良と相関することが判明した(後述の実施例を参照)。この知見に基づき本発明の第2の局面は、RFP遺伝子の発現量を利用した抗がん剤耐性検査法(以下、「本発明の検査法」と称する)を提供する。本発明の検査法を行うと、がん細胞の抗がん剤耐性を判定することができる。判定結果は例えば治療方針の決定(効果的な治療法の選択など)に利用される。判定結果を利用することによって治療成績の向上、予後改善、患者の生活の質(QOL)の向上などがもたらされる。このように本発明の検査法は、がん治療に極めて有用な情報を提供する。
免疫組織化学ではRFPを特異的に認識する抗体(抗RFP抗体)を使用し、当該抗体の結合性(結合量)を指標としてRFPタンパク質量を調べる。抗RFP抗体を使用した免疫組織化学は例えばABC(Avidin-Biotin Complex)法、PAP(Peroxydase-anti-Peroxydase Complex)法、LAB(Linked Avidin-Biotin)法、LSAB(Linked Streptavidin-Biotin)法等で行うことができる。これらの各方法についての標準的なプロコールは周知である(例えば、「酵素抗体法、改訂第3版」、渡辺慶一、中根一穂編集、学際企画を参照)。
(1)固定・パラフィン包埋
生体(剖検症例の場合は死体)より採取した組織をホルマリンやパラフォルムアルデヒド等によって固定する。その後パラフィン包埋する。一般にアルコールで脱水した後キシレンで処理し、最後にパラフィンで包埋する。パラフィンで包埋された標本を所望の厚さ(例えば3~5μm)に薄切し、スライドガラス上に伸展させる。尚、パラフィン包埋標本に代えて凍結標本を用いる場合もある。
(2)脱パラフィン
一般にキシレン、アルコール、及び精製水で順に処理する。
(3)前処理(抗原賦活)
必要に応じて抗原賦活のために酵素処理、加熱処理及び/又は加圧処理等を行う。
(4)内因性ペルオキシダーゼ除去
染色の際の標識物質としてペルオキシダーゼを使用する場合、過酸化水素水で処理して内因性ペルオキシダーゼ活性を除去しておく。
(5)非特異的反応阻害
切片をウシ血清アルブミン溶液(例えば1%溶液)で数分から数十分程度処理して非特異的反応を阻害する。尚、ウシ血清アルブミンを含有させた抗体溶液を使用して次の一次抗体反応を行うこととし、この工程を省略してもよい。
(6)一次抗体反応
適当な濃度に希釈した抗体をスライドガラス上の切片に滴下し、その後数十分~数時間反応させる。反応終了後、リン酸緩衝液など適当な緩衝液で洗浄する。
(7)標識試薬の添加
標識物質としてペルオキシダーゼが頻用される。ペルオキシダーゼを結合させた2次抗体をスライドガラス上の切片に滴下し、その後数十分~数時間反応させる。反応終了後、リン酸緩衝液など適当な緩衝液で洗浄する。
(8)発色反応
トリス緩衝液にDAB(3,3'-diaminobenzidine)を溶解する。続いて過酸化水素水を添加する。このようにして調製した発色用溶液を数分間(例えば5分間)切片に浸透させ、発色させる。発色後、切片を水道水で十分に洗浄し、DABを除去する。
(9)核染色
マイヤーのヘマトキシリンを数秒~数十秒反応させて核染色を行う。流水で洗浄し色出しする(通常、数分間)。
(10)脱水、透徹、封入
アルコールで脱水した後、キシレンで透徹処理し、最後に合成樹脂やグリセリン、ゴムシロップなどで封入する。
当業者であればRFPの配列(NCBIのデータベース、Accession: RFPNM_006510、DEFINITION: Homo sapiens tripartite motif-containing 27 (TRIM27), mRNA.(配列番号2))を基に各方法に適した核酸プライマー又は核酸プローブを常法で設計することができる。
<例1>
区分1:RFP陽性である:抗がん剤耐性である
区分2:RFP陰性である:抗がん剤感受性である
<例2>
区分1:RFPの発現を認めず:抗がん剤感受性である
区分2:RFPの弱い発現を認める:抗がん剤に対する耐性は低い
区分3:RFPの中程度の発現を認める:抗がん剤に対する耐性は中程度である
区分4:RFPの強い発現を認める:抗がん剤に対する耐性は高い
本発明者らの検討の結果、RFPの高発現が大腸癌患者や子宮体がん患者の予後不良と相関することが判明した(後述の実施例を参照)。この知見に基づき本発明の第3の局面は、RFPからなる、がん患者の予後推定用マーカーを提供する。当該バイオマーカーはがん患者の予後を把握する上で有用である。また、術式を決定する際の指針にもなる。例えば、子宮体がんの患者から採取した検体(がん細胞)においてRFPの発現を認めた場合(例えば免疫染色で陽性と判断した場合)、より積極的な術式を採用することが好ましいと判定することが可能である。より積極的な術式としては広範子宮全摘術、リンパ説郭清を挙げることができる。
<例1>
区分1:RFP陽性である:予後が悪い
区分2:RFP陰性である:予後が良い
<例2>
区分1:RFPの発現を認めず:予後が良い
区分2:RFPの弱い発現を認める:予後が比較的良い
区分3:RFPの中程度の発現を認める:予後が比較的悪い
区分4:RFPの強い発現を認める:予後が悪い
1.材料及び方法
(1)プラスミド
pcDNA3-HDAC1-Flagは川口博士(愛知県心身障害者コロニー)より供与された。全長NF-YA、NF-YB、NF-YC、HDAC1、HDAC1欠失断片、及びTBP-2 cDNAをpcDNA3.1/V5-His-TOPOベクターにクローニングした。操作は取り扱い説明書に従った。pcDNA3.1/V5-His-HDAC1から切り出したHDAC1 cDNAをpcDNA3.1/myc-Hisベクターに挿入した。pFlag-RFPコンストラクトについては記述の通りである(参考文献20)。RFP cDNAをpEGFP-C1ベクターに挿入した。
HDAC1 siRNAはDhamacon社から、siRFP, siTBP-2, siNF-YCはQIAGEN社から購入した。siRNAはLipofectamine 2000 (Invitrogen)を用いて細胞内に導入した。shRNAを利用したノックダウンのためにRFPを標的とするshRNAをコードする一本鎖オリゴヌクレオチドおよびその相補配列を合成した(以下にセンス鎖を示す)。
shRFP: 5’- GATCGAGTTACTCGGGAGGGAAATTCAAGAGATTTCCCTCCCGAGTAACTCTTTTTTGGAAA -3’(配列番号10)
この相補的なオリゴヌクレオチド対をpSilencer3.1-H1 neo vector (Ambion)に挿入し、shRNA発現ベクターとして用いた。
HeLa細胞に上記のshRNA発現ベクターをLipofectamine 2000を用いて導入し、ネオマイシンで2週間選択を行った。
8 x 103の細胞を96穴プレートにまき、24時間後にH2O2(10時間)またはシスプラチン(24時間)を記述した濃度で処理を行った。生存率の計測はWST-1法(Roche)を用いて行った。グラフは未処理の細胞の値を1とした相対値で示している。
siRNAでトランスフェクトしたHeLa細胞からRNeasy Mini Kit (キアゲン)を用いて全RNAを調製した。Superscript II (インビトロジェン)を用いてcDNA転写物を調製した。RT-PCRにはHDAC1特異的プライマー(センス:5’- CTCCTGTTTTTTTCAGGCTCC-3’(配列番号11)、アンチセンス:5’- AGGAGAAGACAGACAGAGGGC-3’(配列番号12))、RFP特異的プライマー(センス:5’-TGCTCGACTGCGGCCATAAC-3’(配列番号13)、アンチセンス:5’-TCGGTGCGCAGCTGCTTTAC-3’(配列番号14))、TBP-2特異的プライマー(センス:5’- TGAGATGGTGATCATGAGACC -3’(配列番号15)、アンチセンス:5’- GTATTGACATCCACCAGATCC -3’(配列番号16))GAPDH特異的プライマー(センス:5’-GAAGGTGAAGGTCGGAGTCAA-3’(配列番号17)、アンチセンス:5’-GAGATGATGACCCTTTTGGCTC-3’(配列番号18))を用いた。
細胞を氷冷PBSで2回洗浄した後、プロテアーゼインヒビターを含有する溶解バッファー(50 mM Tris-HCl, pH 7.4, 120 mM NaCl, 5 mM MgCl2, 0.8% Nonidet P-40, 10% glycerol, 1mM DTT and 1 mM PMSF)で溶解した。ライセートを超音波処理した後、各抗体で免疫沈降した。精製した正常ウサギ抗体又は抗HAタグ抗体をコントロールに用いた。電気泳動による分離及びウエスタンブロットは既述の方法(参考文献20)に従った。ウサギポリクローナル抗体を用いたウエスタンブロットにはRelia Blot(Bethyl Laboratories)を使用した。
(7)クロマチン免疫沈降(ChIP)アッセイ
既述の方法に従ってクロマチン免疫沈降アッセイを行った(参考文献28)。簡単に説明すると、ホルムアルデヒドで架橋したクロマチンを超音波処理し、得られたクロマチン断片を抗HDAC1抗体、抗アセチル化H3抗体、抗アセチル化H4抗体、抗RFP抗体又は抗NFYB抗体で免疫沈降した。精製した正常ウサギ又はマウスIgGをコントロールに用いた。5M NaClを終濃度で0.2Mとなるように添加した後、65℃で8時間インキュベートして脱架橋した。精製DNA及び全細胞DNAをPCR(32~37サイクル)で増幅した。
コントロールもしくはRFPのshRNAの安定発現株を、7週齢のメスのヌードマウスの背部に移植した。腫瘍の体積が50mm3に達した時点でマウスをシスプラチン治療群と無治療群に無作為に分けた。用量1mg/kgのシスプラチンもしくは溶媒(生理食塩水:コントロール)を4日毎にマウスの腹腔内に投与した。腫瘍の体積は、ノギスを用いて腫瘍径を測定し、以下の式で計算した。
体積=(長径 x 短径 x 短径)/2
今回の実験では、ホルマリンで固定し、パラフィン包埋した大腸癌の組織切片を用いた。標本をキシレン中で脱パラフィン処理し、段階的に希釈したエタノールに順次浸けることで親水化させた。Protein Blocking Agent(UltraTech HRP Streptavidin-Biotin Detection System, Beckman Coulter)を用いて非特異的なシグナルの抑制処理を行った。さらに、3%過酸化水素水で内因性ペルオキシダーゼの不活性化処理を行った。0.5%NP-40を含むクエン酸緩衝液(pH7.0)中に浸け、121℃、10分間オートクレーブをかけて抗原の賦活化を行った。サンプルを抗RFPウサギポリクローナル抗体と反応させた後に、ビオチン化したヤギの二次抗体を反応させた。さらに、ペルオキシダーゼを付加したストレプトアビジンを反応させ、3, 3′-diaminobenzidine tetrahydrochloride(DAB)を用いて発色させた。茶色の染色が10%以上の細胞で見られる標本をRFP陽性と判定した。
統計解析ソフトStat Viewを用いて、Kaplan-Meier法によってRFPの発現と大腸癌患者の予後との関連性を検討した。
上述の大腸癌組織標本をキシレン中で脱パラフィン処理をし、段階的に希釈したエタノールに順次浸けることで親水化させた。クエン酸緩衝液(pH6.0)中で10分間マイクロウェーブ処理をして抗原を賦活化させた。サンプルを1%BSAで処理することで非特異的なシグナルの検出を抑制した。サンプルに抗RFPウサギポリクローナル抗体および抗TBP-2マウスモノクローナル抗体を反応させた後、Alexa Fluor 594を付加した抗ウサギIgG抗体およびAlexa Fluor 488を付加した抗マウスIgG抗体を反応させた。細胞核を、DAPIを用いて染色した。スライドにマウント剤を乗せて標本とし、共焦点顕微鏡を用いて観察及び撮影を行った。
1992~2007年の間に名古屋大学病院で治療を受けた子宮体がん患者から採取した試料(119検体)をインフォームドコンセントの下、実験に用いた。患者の年齢は28~86歳である(平均年齢57歳)。ネオアジュバンド化学療法を受けた患者は含まれていない。全ての患者は、子宮全摘出術又は広範子宮全摘術と両側卵管卵巣摘出術のいずれかを経験している。また、72名の患者にはリンパ節郭清が行われている。ステージ1998年のFigO(International Federation of Gynecology and Obstetrics)基準に従って各患者のステージを決定した。ステージIが72名、ステージIIが16名は、ステージIIIが24名、ステージIVが7名であった。組織学的グレードについては世界保健機構の基準に従った。G1が50名、G2が51名、G3が18名であった。
初めに、HDACiがHeLa細胞の酸化ストレスに対する感受性に影響を与えるかどうかを検討した。代表的なHDACiであるTrichostatin A (TSA)によって処理したHeLa細胞では、酸化ストレスの誘導剤であるH2O2に対する感受性が亢進していることを見出した(図1a)。このHDACiによる感受性の亢進に関わる分子機構を解明するため、HDAC1に着目して研究を進めた。HDAC1をsiRNAでノックダウンすると、H2O2およびシスプラチンの細胞障害作用が著しく亢進した(図1b~d)。このことから、HDACiが、HDAC1の機能阻害を介して酸化ストレス感受性を亢進させている可能性が示唆された。HDAC1によるストレス感受性の制御の分子機構に関しては、p53との相互作用を介したものが推測されるが(参考文献16~19)、今回用いたHeLa細胞ではパピローマウイルスのE6タンパク質によってp53の発現が抑制されているため、他の機構を介して制御されている可能性が高い。
さらに、同様の結果が大腸癌および乳がんの細胞株を用いた際にも観察されることを確認した(図4)。
今回の発見は、HDACiと抗がん剤の相乗効果における分子機構に新たな知見をもたらすものである。
本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
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Claims (15)
- 酸化ストレス誘導能を有する抗がん剤の作用増強剤であって、RFP(RET finger protein)遺伝子の発現又はRFPの作用を抑制する化合物を有効成分とする作用増強剤。
- 前記化合物が以下の(a)~(d)からなる群より選択される化合物である、請求項1に記載の作用増強剤:
(a)RFP遺伝子を標的とするsiRNA;
(b)RFP遺伝子を標的とするsiRNAを細胞内で生成する核酸コンストラクト;
(c)RFP遺伝子の転写産物を標的とするアンチセンス核酸;
(d)RFP遺伝子の転写産物を標的とするリボザイム。 - 酸化ストレス誘導能を有する抗がん剤と併用される、請求項1又は2に記載の作用増強剤。
- 酸化ストレス誘導能を有する抗がん剤の作用を増強する方法であって、標的がん細胞内において、RFP遺伝子の発現又はRFPの作用を抑制するステップを含む方法。
- 請求項1又は2に記載の作用増強剤を投与するステップと、酸化ストレス誘導能を有する抗がん剤を投与するステップと、を含むがん治療法。
- 生体から分離されたがん細胞内のRFP発現量を調べるステップと、
前記ステップの結果に基づき、酸化ストレス誘導能を有する抗癌剤に対する前記がん細胞の耐性を判定するステップと、を含む抗がん剤耐性検査法。 - RFPからなる、がん患者の予後推定用バイオマーカー。
- がんが大腸癌又は子宮体がんである、請求項7に記載の予後推定用バイオマーカー。
- がん患者から分離されたがん細胞内のRFP発現量を指標とした、がん患者の予後推定法。
- がん細胞内のRFP発現量が多いことが、予後が不良であることを示す、請求項9に記載の予後推定法。
- がんが大腸癌又は子宮体がんである、請求項9又は10に記載の予後推定法。
- 抗RFP抗体からなる、がん患者の予後推定用試薬。
- がんが大腸癌又は子宮体がんである、請求項12に記載の予後推定用試薬。
- 請求項12又は13に記載の試薬を含む、がん患者の予後推定用キット。
- がんが大腸癌又は子宮体がんである、請求項14に記載の予後推定用キット。
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WO2004066941A2 (en) * | 2003-01-24 | 2004-08-12 | Bayer Pharmaceuticals Corporation | Expression profiles for colon cancer and methods of use |
CA2528669A1 (en) | 2003-06-09 | 2005-01-20 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
CA2475769C (en) * | 2003-08-28 | 2018-12-11 | Veridex, Llc | Colorectal cancer prognostics |
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JP5682975B2 (ja) * | 2010-09-02 | 2015-03-11 | 国立大学法人東北大学 | がん発症又はがん発症リスクの判定方法 |
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JP2014112096A (ja) | 2014-06-19 |
ES2432537T3 (es) | 2013-12-04 |
US20130177660A1 (en) | 2013-07-11 |
EP2322224A4 (en) | 2012-02-29 |
US8936906B2 (en) | 2015-01-20 |
EP2322224A1 (en) | 2011-05-18 |
CN104698168A (zh) | 2015-06-10 |
EP2322224B1 (en) | 2013-07-17 |
JPWO2010023917A1 (ja) | 2012-01-26 |
JP5590693B2 (ja) | 2014-09-17 |
JP5515071B2 (ja) | 2014-06-11 |
US20110160287A1 (en) | 2011-06-30 |
US8372583B2 (en) | 2013-02-12 |
CN102131525A (zh) | 2011-07-20 |
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