JP2010259353A - Method for predicting the effectiveness of 5-aminolevulinic acid - Google Patents
Method for predicting the effectiveness of 5-aminolevulinic acid Download PDFInfo
- Publication number
- JP2010259353A JP2010259353A JP2009111948A JP2009111948A JP2010259353A JP 2010259353 A JP2010259353 A JP 2010259353A JP 2009111948 A JP2009111948 A JP 2009111948A JP 2009111948 A JP2009111948 A JP 2009111948A JP 2010259353 A JP2010259353 A JP 2010259353A
- Authority
- JP
- Japan
- Prior art keywords
- ala
- pept1
- gene
- pdt
- tumor suppressor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 100
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 229960002749 aminolevulinic acid Drugs 0.000 title claims abstract description 48
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 52
- 201000011510 cancer Diseases 0.000 claims abstract description 42
- 108091006594 SLC15A1 Proteins 0.000 claims abstract description 40
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 claims abstract description 28
- 102000015098 Tumor Suppressor Protein p53 Human genes 0.000 claims abstract description 26
- 206010064571 Gene mutation Diseases 0.000 claims abstract description 18
- 238000002428 photodynamic therapy Methods 0.000 claims abstract description 14
- 102000046014 Peptide Transporter 1 Human genes 0.000 claims abstract description 12
- 239000012472 biological sample Substances 0.000 claims abstract description 11
- 238000003745 diagnosis Methods 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 33
- 108020004705 Codon Proteins 0.000 claims description 22
- 108020004999 messenger RNA Proteins 0.000 claims description 17
- 239000000523 sample Substances 0.000 claims description 17
- 101150088063 pept-1 gene Proteins 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 238000002509 fluorescent in situ hybridization Methods 0.000 claims description 10
- 238000009396 hybridization Methods 0.000 claims description 8
- 238000003757 reverse transcription PCR Methods 0.000 claims description 8
- 238000012163 sequencing technique Methods 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 238000001262 western blot Methods 0.000 claims description 7
- 238000002965 ELISA Methods 0.000 claims description 5
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 claims description 5
- 238000003753 real-time PCR Methods 0.000 claims description 4
- WJXSXWBOZMVFPJ-NENRSDFPSA-N N-[(2R,3R,4R,5S,6R)-4,5-dihydroxy-6-methoxy-2,4-dimethyloxan-3-yl]-N-methylacetamide Chemical compound CO[C@@H]1O[C@H](C)[C@@H](N(C)C(C)=O)[C@@](C)(O)[C@@H]1O WJXSXWBOZMVFPJ-NENRSDFPSA-N 0.000 claims description 3
- 241000718541 Tetragastris balsamifera Species 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 claims description 3
- 238000001727 in vivo Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 58
- 102100021491 Solute carrier family 15 member 1 Human genes 0.000 description 28
- 230000035772 mutation Effects 0.000 description 23
- 108020004414 DNA Proteins 0.000 description 22
- 239000000243 solution Substances 0.000 description 18
- 210000001519 tissue Anatomy 0.000 description 14
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 239000013615 primer Substances 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 208000005718 Stomach Neoplasms Diseases 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 206010017758 gastric cancer Diseases 0.000 description 8
- 230000002441 reversible effect Effects 0.000 description 8
- 201000011549 stomach cancer Diseases 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- ZLHFONARZHCSET-UHFFFAOYSA-N 5-aminolevulinic acid hydrochloride Chemical compound Cl.NCC(=O)CCC(O)=O ZLHFONARZHCSET-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 6
- 244000172533 Viola sororia Species 0.000 description 6
- 239000013078 crystal Substances 0.000 description 6
- 108700025694 p53 Genes Proteins 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 239000013076 target substance Substances 0.000 description 6
- 239000002033 PVDF binder Substances 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- -1 etc.) Chemical compound 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- HAGVCKULCLQGRF-UHFFFAOYSA-N pifithrin Chemical compound [Br-].C1=CC(C)=CC=C1C(=O)CN1[C+](N)SC2=C1CCCC2 HAGVCKULCLQGRF-UHFFFAOYSA-N 0.000 description 5
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 125000004494 ethyl ester group Chemical group 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 125000004492 methyl ester group Chemical group 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 206010009944 Colon cancer Diseases 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- 230000037429 base substitution Effects 0.000 description 3
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 229960003964 deoxycholic acid Drugs 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 230000026731 phosphorylation Effects 0.000 description 3
- 238000006366 phosphorylation reaction Methods 0.000 description 3
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- UZFPOOOQHWICKY-UHFFFAOYSA-N 3-[13-[1-[1-[8,12-bis(2-carboxyethyl)-17-(1-hydroxyethyl)-3,7,13,18-tetramethyl-21,24-dihydroporphyrin-2-yl]ethoxy]ethyl]-18-(2-carboxyethyl)-8-(1-hydroxyethyl)-3,7,12,17-tetramethyl-22,23-dihydroporphyrin-2-yl]propanoic acid Chemical compound N1C(C=C2C(=C(CCC(O)=O)C(C=C3C(=C(C)C(C=C4N5)=N3)CCC(O)=O)=N2)C)=C(C)C(C(C)O)=C1C=C5C(C)=C4C(C)OC(C)C1=C(N2)C=C(N3)C(C)=C(C(O)C)C3=CC(C(C)=C3CCC(O)=O)=NC3=CC(C(CCC(O)=O)=C3C)=NC3=CC2=C1C UZFPOOOQHWICKY-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- KSFOVUSSGSKXFI-GAQDCDSVSA-N CC1=C/2NC(\C=C3/N=C(/C=C4\N\C(=C/C5=N/C(=C\2)/C(C=C)=C5C)C(C=C)=C4C)C(C)=C3CCC(O)=O)=C1CCC(O)=O Chemical compound CC1=C/2NC(\C=C3/N=C(/C=C4\N\C(=C/C5=N/C(=C\2)/C(C=C)=C5C)C(C=C)=C4C)C(C)=C3CCC(O)=O)=C1CCC(O)=O KSFOVUSSGSKXFI-GAQDCDSVSA-N 0.000 description 2
- VSEIDZLLWQQJGK-CHOZPQDDSA-N CCC1=C(C)C2=N\C\1=C/C1=C(C)C(C(O)=O)=C(N1)\C(CC(=O)N[C@@H](CC(O)=O)C(O)=O)=C1/N=C(/C=C3\N/C(=C\2)C(C=C)=C3C)[C@@H](C)[C@@H]1CCC(O)=O Chemical compound CCC1=C(C)C2=N\C\1=C/C1=C(C)C(C(O)=O)=C(N1)\C(CC(=O)N[C@@H](CC(O)=O)C(O)=O)=C1/N=C(/C=C3\N/C(=C\2)C(C=C)=C3C)[C@@H](C)[C@@H]1CCC(O)=O VSEIDZLLWQQJGK-CHOZPQDDSA-N 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 230000004568 DNA-binding Effects 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 101100532414 Homo sapiens SLC15A1 gene Proteins 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 108091006593 SLC15A2 Proteins 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 102100021488 Solute carrier family 15 member 2 Human genes 0.000 description 2
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 2
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 208000035269 cancer or benign tumor Diseases 0.000 description 2
- 230000022534 cell killing Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 229930002875 chlorophyll Natural products 0.000 description 2
- 235000019804 chlorophyll Nutrition 0.000 description 2
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 150000003278 haem Chemical class 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 101150016475 pept-2 gene Proteins 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 230000000865 phosphorylative effect Effects 0.000 description 2
- 230000002165 photosensitisation Effects 0.000 description 2
- 229960004293 porfimer sodium Drugs 0.000 description 2
- 150000004032 porphyrins Chemical class 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 229950003776 protoporphyrin Drugs 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229950010924 talaporfin Drugs 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 239000000225 tumor suppressor protein Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 206010001497 Agitation Diseases 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010003594 Ataxia telangiectasia Diseases 0.000 description 1
- 206010003645 Atopy Diseases 0.000 description 1
- VGGGPCQERPFHOB-MCIONIFRSA-N Bestatin Chemical compound CC(C)C[C@H](C(O)=O)NC(=O)[C@@H](O)[C@H](N)CC1=CC=CC=C1 VGGGPCQERPFHOB-MCIONIFRSA-N 0.000 description 1
- VGGGPCQERPFHOB-UHFFFAOYSA-N Bestatin Natural products CC(C)CC(C(O)=O)NC(=O)C(O)C(N)CC1=CC=CC=C1 VGGGPCQERPFHOB-UHFFFAOYSA-N 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 1
- UJKPHYRXOLRVJJ-MLSVHJFASA-N CC(O)C1=C(C)/C2=C/C3=N/C(=C\C4=C(CCC(O)=O)C(C)=C(N4)/C=C4\N=C(\C=C\1/N\2)C(C)=C4C(C)O)/C(CCC(O)=O)=C3C Chemical class CC(O)C1=C(C)/C2=C/C3=N/C(=C\C4=C(CCC(O)=O)C(C)=C(N4)/C=C4\N=C(\C=C\1/N\2)C(C)=C4C(C)O)/C(CCC(O)=O)=C3C UJKPHYRXOLRVJJ-MLSVHJFASA-N 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 206010008263 Cervical dysplasia Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 208000005590 Choroidal Neovascularization Diseases 0.000 description 1
- 206010060823 Choroidal neovascularisation Diseases 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 235000005956 Cosmos caudatus Nutrition 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- 230000007035 DNA breakage Effects 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 101710200526 DNA polymerase 2 Proteins 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 102000012199 E3 ubiquitin-protein ligase Mdm2 Human genes 0.000 description 1
- 108050002772 E3 ubiquitin-protein ligase Mdm2 Proteins 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- AEMRFAOFKBGASW-UHFFFAOYSA-M Glycolate Chemical compound OCC([O-])=O AEMRFAOFKBGASW-UHFFFAOYSA-M 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 201000011062 Li-Fraumeni syndrome Diseases 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000005228 Pericardial Effusion Diseases 0.000 description 1
- 206010034972 Photosensitivity reaction Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 206010038111 Recurrent cancer Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 description 1
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- HDOVUKNUBWVHOX-QMMMGPOBSA-N Valacyclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCOC(=O)[C@@H](N)C(C)C)C=N2 HDOVUKNUBWVHOX-QMMMGPOBSA-N 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
- 235000013487 Viola odorata Nutrition 0.000 description 1
- 235000002254 Viola papilionacea Nutrition 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 206010064930 age-related macular degeneration Diseases 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 125000005210 alkyl ammonium group Chemical group 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000005546 dideoxynucleotide Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- ZFKJVJIDPQDDFY-UHFFFAOYSA-N fluorescamine Chemical compound C12=CC=CC=C2C(=O)OC1(C1=O)OC=C1C1=CC=CC=C1 ZFKJVJIDPQDDFY-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 238000003505 heat denaturation Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 108010028721 hydrogen-coupled oligopeptide transporter PepT2 Proteins 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 229940049920 malate Drugs 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Chemical class 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 201000011591 microinvasive gastric cancer Diseases 0.000 description 1
- 210000000110 microvilli Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000036438 mutation frequency Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 108010082406 peptide permease Proteins 0.000 description 1
- 210000004912 pericardial fluid Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229940109328 photofrin Drugs 0.000 description 1
- 230000036211 photosensitivity Effects 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 125000001325 propanoyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000022983 regulation of cell cycle Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 229950009811 ubenimex Drugs 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229940093257 valacyclovir Drugs 0.000 description 1
- 229940070710 valerate Drugs 0.000 description 1
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- ZQFGRJWRSLZCSQ-ZSFNYQMMSA-N verteporfin Chemical compound C=1C([C@@]2([C@H](C(=O)OC)C(=CC=C22)C(=O)OC)C)=NC2=CC(C(=C2C=C)C)=NC2=CC(C(=C2CCC(O)=O)C)=NC2=CC2=NC=1C(C)=C2CCC(=O)OC ZQFGRJWRSLZCSQ-ZSFNYQMMSA-N 0.000 description 1
- 229960003895 verteporfin Drugs 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
【課題】５−アミノレブリン酸−光線力学的療法（ＡＬＡ−ＰＤＴ）又は５−アミノレブリン酸−光線力学的診断（ＡＬＡ−ＰＤＤ）の施術前に、生体内に投与する５−アミノレブリン酸（ＡＬＡ）の有効性の判定方法、及びそれらのためのキットを提供すること。
【解決手段】がん患者から採取した生体試料における、ＰＥＰＴ１（ペプチドトランスポーター１）の発現量が多い場合、また、がん抑制タンパク質ｐ５３の遺伝子変異を検出する場合に、５−アミノレブリン酸（ＡＬＡ）類を投与することが、ＡＬＡ−ＰＤＴ又はＡＬＡ−ＰＤＤに有効であることを確認した。
【選択図】なしBefore the treatment of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT) or 5-aminolevulinic acid-photodynamic diagnosis (ALA-PDD), 5-aminolevulinic acid (ALA) is administered in vivo. To provide a method for determining efficacy, and a kit for them.
When the expression level of PEPT1 (peptide transporter 1) is high in a biological sample collected from a cancer patient, or when detecting a gene mutation of the tumor suppressor protein p53, 5-aminolevulinic acid (ALA) is used. It was confirmed that administration of) was effective for ALA-PDT or ALA-PDD.
[Selection figure] None
Description
本発明は、５−アミノレブリン酸類投与による光線力学治療の有効性を手術前に判断しうる有効性予測方法に関し、さらに詳しくは、５−アミノレブリン酸類投与後のＰＥＰＴ１発現とｐ５３の変異の有無を確認することを特徴とする５−アミノレブリン酸−光線力学的療法や５−アミノレブリン酸−光線力学的診断の手術前有効性予測方法に関する。 The present invention relates to a method for predicting the effectiveness of photodynamic therapy by administration of 5-aminolevulinic acids before surgery, and more specifically, confirming the presence of PEPT1 expression and p53 mutation after administration of 5-aminolevulinic acids The present invention relates to a method for predicting preoperative effectiveness of 5-aminolevulinic acid-photodynamic therapy and 5-aminolevulinic acid-photodynamic diagnosis.
日本国内のがんによる死亡率の年次推移は、２０年前から大きな変化が見られていない。このことは、現在の治療だけでは、がんを克服することが困難であることを示している。がんという疾患を克服するためには、全身に存在するがん細胞や目に見えない微小ながんの転移に適用可能で、進行がん（再発がん、転移がん）を食い止める必要があるが、今後高齢化社会を迎えるにあたり、生活の質（Quality of Life：ＱＯＬ）を高いレベルで維持できる新しい治療技術の普及が急務であり、その要請はますます強くなると考えられる。 The annual trend of cancer mortality in Japan has not changed significantly since 20 years ago. This indicates that it is difficult to overcome cancer with current treatment alone. In order to overcome the disease called cancer, it can be applied to metastasis of cancer cells that exist throughout the body and invisible small cancers, and it is necessary to stop advanced cancer (recurrent cancer, metastatic cancer) However, as we enter an aging society in the future, there is an urgent need to spread new treatment techniques that can maintain a high quality of life (Quality of Life: QOL), and the demand is expected to become stronger.
光線力学的療法（Photodynamic Therapy：以下「ＰＤＴ」ともいう。）は、光に反応する化合物を投与し、光を照射することにより標的箇所を治療する方法であり、治療が簡便で、生体侵襲性が小さく、臓器温存が可能であることなどから、近年、ＱＯＬを考慮した新たながん治療法として注目されている。ＰＤＴは、投与されたポルフィリン関連化合物が直接腫瘍組織や新生血管へ特異的に集積した後、レーザー光などの光の励起により生ずる一重項酸素が細胞を変性、壊死に陥らせることにより治療効果を発揮するものである。 Photodynamic therapy (hereinafter also referred to as “PDT”) is a method of treating a target site by administering a compound that reacts with light and irradiating light, and is easy to treat and bioinvasive. In recent years, it has attracted attention as a new cancer treatment method considering QOL. In PDT, administered porphyrin-related compounds are directly accumulated in tumor tissues and new blood vessels, and then singlet oxygen generated by excitation of light such as laser light causes the cells to degenerate and become necrotic. It is something that demonstrates.
現在、ＰＤＴは、早期肺がん（病期０期又は病期Ｉ期肺がん），表在型早期胃がん，表在型食道がん，子宮頸部初期がん及び異形成に対して保険適用されている。がん治療を目的に国内で承認されている光増感剤にはヘマトポルフィリン誘導体のポルフィマーナトリウム（商品名：フォトフリン）や、植物クロロフィル由来のタラポルフィンナトリウム（商品名：レザフィリン）がある。ポルフィマーナトリウムは代謝が遅く、表皮組織付近に集積しやすい性質を持っているため、日光過敏症をおこす。患者は治療後約１ヵ月間、遮光された環境で生活する必要がある。タラポルフィンナトリウムは皮膚への残留性が低く、日光過敏症がより軽度で、組織透過性のよりよい長波長側(６６４ｎｍ)でのレーザー光で励起可能であるという特徴を有している。その他、がん治療以外にも、ベルテポルフィン(ビスダイン)が滲出型加齢性黄班変性症による脈絡膜新生血管に対して優れた治療効果を持つことが証明され、国内においては２００３年１０月に承認されている。 Currently, PDT is insured against early lung cancer (stage 0 or stage I lung cancer), superficial early gastric cancer, superficial esophageal cancer, early cervical cancer, and dysplasia. . Photosensitizers approved in Japan for cancer treatment include hematoporphyrin derivative porfimer sodium (trade name: Photofrin) and talaporfin sodium derived from plant chlorophyll (trade name: Rezaphyrin). Porfimer sodium has a slow metabolism and tends to accumulate in the vicinity of the epidermal tissue, so it causes sunlight hypersensitivity. Patients need to live in a shaded environment for about a month after treatment. Talaporfin sodium is characterized by low persistence on the skin, milder photosensitivity, and excitability with laser light on the longer wavelength side (664 nm) with better tissue permeability. In addition to cancer treatment, verteporfin (bisdyne) has been proven to have an excellent therapeutic effect on choroidal neovascularization due to exudative age-related macular degeneration, and in Japan in October 2003 Approved.
また、５−アミノレブリン酸（以下「ＡＬＡ」ともいう）は動物や植物や菌類に広く存在する、生体内に含まれる天然アミノ酸の一種であり、クロロフィルやヘムの共通前駆体として細胞内で代謝を受ける。ＡＬＡは他のポルフィリン関連化合物と同様に腫瘍組織に選択的に集積する性質を有するが、それ自体には光感受性はなく、細胞内でヘム生合成経路の一連の酵素群によりプロトポルフィリンＩＸ（以下「ＰｐＩＸ」ともいう）に代謝活性化され、がん細胞内で蓄積した後に、光線を照射すると細胞内で一重項酸素を生じ、細胞を変成・壊死させることができるとされており、５−アミノレブリン酸−光線力学的療法（以下「ＡＬＡ−ＰＤＴ」ともいう）の研究が進められている。ＰｐＩＸは４０５ｎｍの励起光を受けると、６３５ｎｍにピークを持つ赤色蛍光を発することから、光線力学的診断（Photodynamic diagnosis：以下「ＰＤＤ」ともいう。）による腫瘍の診断において、ＡＬＡを用いる５−アミノレブリン酸−光線力学的診断（以下「ＡＬＡ−ＰＤＤ」ともいう、特許文献１参照）への応用が可能であり、現在、ＡＬＡは脳腫瘍や膀胱がんの診断、貧血予防やアトピー対策などでの用途が期待されている。 5-Aminolevulinic acid (hereinafter also referred to as “ALA”) is a kind of natural amino acid that is widely present in animals, plants, and fungi, and is metabolized in cells as a common precursor of chlorophyll and heme. receive. ALA, like other porphyrin-related compounds, has the property of selectively accumulating in tumor tissue, but itself is not photosensitive and protoporphyrin IX (hereinafter referred to as “protoporphyrin IX”) by a series of enzymes in the heme biosynthetic pathway in cells It is said that when it is irradiated with light after being metabolically activated and accumulated in cancer cells, singlet oxygen is generated in the cells, and the cells can be metamorphosed and necrotized. Studies on aminolevulinic acid-photodynamic therapy (hereinafter also referred to as “ALA-PDT”) are in progress. When PpIX receives excitation light of 405 nm, PpIX emits red fluorescence having a peak at 635 nm, so that 5-aminolevulin using ALA is used in tumor diagnosis by photodynamic diagnosis (hereinafter also referred to as “PDD”). It can be applied to acid-photodynamic diagnosis (hereinafter also referred to as “ALA-PDD”, see Patent Document 1). Currently, ALA is used for diagnosis of brain tumors and bladder cancer, anemia prevention, atopy measures, etc. Is expected.
一方、ペプチドトランスポーター１（以下「ＰＥＰＴ１」ともいう）は、１９９４年以降ウサギ、ヒト及びラットの小腸からクローニングされ（例えば、非特許文献１〜４参照）、ＰＥＰＴ１を介した輸送研究が急速に発展してきた。ヒトＰＥＰＴ１は、小腸上皮細胞の刷子縁膜側に高い発現が認められるプロトン共輸送型ペプチドトランスポーターであり（例えば、非特許文献５参照）、小腸からの栄養吸収のために短鎖ペプチドを輸送するのみならず、βラクタム抗生物質、抗がん剤ベスタチン、抗ウイルス薬バラシクロビルなど、多様な薬剤の消化管吸収に大きな役割を果たしていることで注目を集めている。 On the other hand, peptide transporter 1 (hereinafter also referred to as “PEPT1”) has been cloned from the small intestine of rabbits, humans and rats since 1994 (see, for example, Non-Patent Documents 1 to 4), and transport research via PEPT1 has been rapidly conducted. Has developed. Human PEPT1 is a proton co-transport peptide transporter that is highly expressed on the brush border membrane side of small intestinal epithelial cells (see, for example, Non-Patent Document 5), and transports short peptides for nutrient absorption from the small intestine. Not only that, it is attracting attention because it plays a major role in the digestive tract absorption of various drugs such as β-lactam antibiotics, anticancer drug bestatin, and antiviral drug valaciclovir.
他方、ｐ５３は全長３９３アミノ酸からなり、Ｎ末端ドメイン、コアドメイン、Ｃ末端ドメインの３つの領域から構成されているペプチドであり、コアドメインはＤＮＡ結合に関与する領域で、がんに認められる変異のほとんどがこの領域に集中していることが知られている。ｐ５３タンパク質は多彩な活性によって遺伝子の異常から生体を守る機能を担っており、その主な活性としては、遺伝子に異常が発生した細胞における、遺伝子転写制御を介した細胞周期進行の制御・遺伝子修復酵素の活性化・アポトーシス誘導能等を挙げることができる。ｐ５３遺伝子自体に突然変異が生じるとこれらのｐ５３タンパク質の機能が欠損し、腫瘍の発生に至るというメカニズムが考えられている。ヒトがん細胞におけるｐ５３遺伝子の変異は、大腸・胃・乳腺・肺・脳・食道など多くのヒトの腫瘍においてｐ５３遺伝子が突然変異を起こしていることが見い出され、変異したｐ５３タンパク質の異常な蓄積が多くの腫瘍組織において観察されている。さらにがん高発家系においてｐ５３遺伝子に変異が見られることが報告されている(Li-Fraumeni症候群)。また、ＤＮＡに損傷が起こると、リン酸化酵素であるＡＴＭ（ataxia telangiectasia, mutated）が活性化されて、ｐ５３のセリン１５をリン酸化することが知られている。 On the other hand, p53 is a peptide consisting of 393 amino acids and consisting of three regions, N-terminal domain, core domain, and C-terminal domain. Core domain is a region involved in DNA binding and is a mutation found in cancer. It is known that most of these are concentrated in this area. The p53 protein is responsible for protecting the body from genetic abnormalities by a variety of activities. Its main activities are control of cell cycle progression and gene repair through gene transcription control in cells with abnormal genes. Enzyme activation / apoptosis induction ability and the like can be mentioned. It is considered that when a mutation occurs in the p53 gene itself, the function of these p53 proteins is lost, leading to tumor development. The mutation of the p53 gene in human cancer cells has been found to be mutated in many human tumors such as large intestine, stomach, mammary gland, lung, brain, and esophagus. Accumulation has been observed in many tumor tissues. Furthermore, it has been reported that mutations are found in the p53 gene in highly cancerous families (Li-Fraumeni syndrome). It is also known that when DNA is damaged, the phosphorylating enzyme ATM (ataxia telangiectasia, mutated) is activated to phosphorylate serine 15 of p53.
上述のように、ＡＬＡ−ＰＤＴ又はＡＬＡ−ＰＤＤについて、広く研究・開発が行われるようになったが、ＡＬＡを生体内に投与しても、がんの種類によっては、がん細胞に取り込まれるＡＬＡ量に差が生じて、がん細胞内でＡＬＡが変換されたＰｐＩＸにレーザー光を照射した場合に、がん細胞を死滅させる効果に差が生じるという問題があった。本発明の課題は、ＡＬＡ−ＰＤＴ又はＡＬＡ−ＰＤＤの施術前にＡＬＡの有効性を予測する方法を提供することにある。 As described above, ALA-PDT or ALA-PDD has been widely researched and developed, but even if ALA is administered in vivo, it is taken into cancer cells depending on the type of cancer. There is a problem in that when the amount of ALA is different and PpIX in which ALA is converted in the cancer cell is irradiated with laser light, the effect of killing the cancer cell is different. An object of the present invention is to provide a method for predicting the effectiveness of ALA before performing ALA-PDT or ALA-PDD.
本発明者らは、上記課題を解決するため、ＡＬＡをがん細胞に取り込む、細胞表面に存在するトランスポーターの発現と、がん抑制タンパク質ｐ５３の変異との関係に注目して鋭意研究を重ねた結果、がん細胞から、ｍＲＮＡを採取し、既に知られているＰＥＰＴ１の塩基配列（ヒトＰＥＰＴ１：GenBank NM_005073、増幅産物４０２ｂｐ）から、以下のオリゴヌクレオチドプライマー： 5’-GAGGGCCAGGAGAAACAAAGAAACAG-3’(forward)(配列番号１)及び5’-CAGGAACATCACCCTCGTAACCATCT-3’(reverse)(配列番号２)を用いて、ＲＴ−ＰＣＲを行うことによって、ＰＥＰＴ１の発現が確認された場合に、ＡＬＡが効率よくがん細胞に取り込まれ、ＡＬＡ−ＰＤＴ及び／又はＡＬＡ−ＰＤＤに効果があることを確認し、また、がん細胞におけるがん抑制タンパク質ｐ５３の遺伝子配列を決定し、がん抑制タンパク質ｐ５３が変異せずに野生型として存在する場合に、ＡＬＡの投与が、ＡＬＡ−ＰＤＴ及び／又はＡＬＡ−ＰＤＤに有効であることを見い出し、本発明を完成するに至った。さらに、本発明者らは、がん細胞におけるｐ５３の１５番目のセリンがリン酸化されている場合に、ＡＬＡの投与がＡＬＡ−ＰＤＴ及び／又はＡＬＡ−ＰＤＤに効果があることを見い出した。本発明はこれら知見に基づいて完成するに至ったものである。 In order to solve the above-mentioned problems, the present inventors have conducted extensive research focusing on the relationship between the expression of transporters existing on the cell surface that incorporate ALA into cancer cells and the mutation of the tumor suppressor protein p53. As a result, mRNA was collected from the cancer cells, and the following oligonucleotide primer: 5′-GAGGGCCAGGAGAAACAAAGAAACAG-3 ′ (forward ) (SEQ ID NO: 1) and 5′-CAGGAACATCACCCTCGTAACCATCT-3 ′ (reverse) (SEQ ID NO: 2) were used to perform RT-PCR, and when the expression of PEPT1 was confirmed, ALA was efficiently cancerous. Confirmed that it is incorporated into cells and has an effect on ALA-PDT and / or ALA-PDD, and gene sequence of tumor suppressor protein p53 in cancer cells In order to complete the present invention, it is found that when the tumor suppressor protein p53 is present as a wild type without mutation, ALA administration is effective for ALA-PDT and / or ALA-PDD. It came. Furthermore, the present inventors have found that administration of ALA is effective for ALA-PDT and / or ALA-PDD when the 15th serine of p53 in cancer cells is phosphorylated. The present invention has been completed based on these findings.
すなわち本発明は、［１］５−アミノレブリン酸−光線力学的療法（ＡＬＡ−ＰＤＴ）又は５−アミノレブリン酸−光線力学的診断（ＡＬＡ−ＰＤＤ）の施術前に５−アミノレブリン酸（ＡＬＡ）類を投与することの有効性を予測する方法であって、がん患者から採取した生体試料における、ＰＥＰＴ１（ペプチドトランスポーター１）の発現量の多寡を指標として、ＰＥＰＴ１の発現量が多いときに、５−アミノレブリン酸（ＡＬＡ）類がＡＬＡ−ＰＤＴ、及び／又は、ＡＬＡ−ＰＤＤに有効であると判定することを特徴とする有効性予測方法や［２］がん抑制タンパク質ｐ５３の遺伝子変異の有無を指標とし、がん抑制タンパク質ｐ５３の遺伝子変異を検出しないときに、ＡＬＡ−ＰＤＴ、及び／又は、ＡＬＡ−ＰＤＤに有効であると判定することを特徴とする、上記［１］記載の有効性予測方法に関する。 In other words, the present invention relates to [1] 5-aminolevulinic acid-photodynamic therapy (ALA-PDT) or 5-aminolevulinic acid-photodynamic diagnosis (ALA-PDD) prior to treatment with 5-aminolevulinic acid (ALA). 5 is a method for predicting the effectiveness of administration, in which when the expression level of PEPT1 is high, the amount of expression level of PEPT1 (peptide transporter 1) in a biological sample collected from a cancer patient is used as an index. -Aminolevulinic acid (ALA) classifying the effectiveness prediction method characterized by determining that it is effective for ALA-PDT and / or ALA-PDD, and the presence or absence of the gene mutation of [2] tumor suppressor protein p53 As an index, when a gene mutation of the tumor suppressor protein p53 is not detected, it is determined to be effective for ALA-PDT and / or ALA-PDD. Characterized by, on the effectiveness prediction method of the above-mentioned [1], wherein.
また本発明は、［３］ＰＥＰＴ１の発現量を、ＰＥＰＴ１遺伝子のｍＲＮＡを検出することにより測定することを特徴とする上記［１］又は［２］記載の有効性予測方法や、［４］ＰＥＰＴ１遺伝子のｍＲＮＡを、ＲＴ−ＰＣＲ法、ＦＩＳＨ法、ノーザンハイブリダイゼーション法、又はリアルタイムＰＣＲ法により検出することを特徴とする上記［３］記載の有効性予測方法や、［５］ＰＥＰＴ１の発現量を、ウエスタンブロット法又はＥＬＩＳＡ法により測定することを特徴とする上記［１］又は［２］記載の有効性予測方法や、［６］がん抑制タンパク質ｐ５３の遺伝子変異を、１１７〜１４２番目のコドン、１７１〜１８１番目のコドン、２３４〜２５８番目のコドン又は２７０〜２８６番目のコドンにおいて検出することを特徴とする上記［２］〜［５］のいずれか記載の有効性予測方法や、［７］がん抑制タンパク質ｐ５３の遺伝子変異を、１７３番目、２４５番目又は２７３番目のコドンにおいて検出することを特徴とする上記［６］記載の有効性予測方法や、［８］がん抑制タンパク質ｐ５３の遺伝子変異を、ダイレクトシークエンシング法、ＭＡＳＡ法、ＰＣＲ−ＤＧＧＥ法、ＤＮＡ ＦＩＳＨ法、ＰＣＲ−ＳＳＣＰ法、ＰＣＲ−ＳＳＯＰ法、ＰＣＲ−ＲＦＬＰ法、又はＤＮＡ−ＦＩＳＨ法により検出することを特徴とする上記［２］〜［７］のいずれか記載の有効性予測方法や、［９］さらに、がん患者から採取した生体試料に、５−アミノレブリン酸（ＡＬＡ）類を投与した後における、ｐｈｏｓｐｈｏ−ｓｅｒ１５ｐ５３タンパク質の発現量の多寡を指標として、ｐｈｏｓｐｈｏ−ｓｅｒ１５ｐ５３タンパク質の発現量が多いときに、ＡＬＡ−ＰＤＴ、及び／又は、ＡＬＡ−ＰＤＤに有効であると判定することを特徴とする上記［１］〜［８］のいずれか記載の有効性予測方法や、［１０］ｐｈｏｓｐｈｏ−ｓｅｒ１５ｐ５３タンパク質を、抗ｐｈｏｓｐｈｏ−ｓｅｒ１５ｐ５３抗体を用いて検出することを特徴とする上記［９］記載の有効性予測方法に関する。 The present invention also provides [3] the method for predicting the efficacy according to [1] or [2] above, wherein the expression level of PEPT1 is measured by detecting mRNA of the PEPT1 gene, and [4] PEPT1. The method according to [3] above, wherein the gene mRNA is detected by RT-PCR, FISH, Northern hybridization, or real-time PCR, and [5] expression level of PEPT1 The efficacy prediction method according to [1] or [2] above, wherein [6] the gene mutation of the tumor suppressor protein p53 is measured by Western blotting or ELISA; , 171 to 181st codon, 234 to 258th codon, or 270 to 286th codon, The efficacy prediction method according to any one of [2] to [5] above, or [7] detecting a gene mutation of the tumor suppressor protein p53 at the 173rd, 245th or 273rd codon, [8] The efficacy prediction method described in [6] above, and [8] the gene mutation of the tumor suppressor protein p53, direct sequencing method, MASA method, PCR-DGGE method, DNA FISH method, PCR-SSCP method, PCR- The efficacy prediction method according to any one of [2] to [7] above, which is detected by the SSOP method, the PCR-RFLP method, or the DNA-FISH method, [9] and further collected from a cancer patient The amount of expression of phospho-ser15p53 protein after administering 5-aminolevulinic acid (ALA) to the biological sample was used as an index. Any one of [1] to [8] above, wherein when the expression level of phospho-ser15p53 protein is large, it is determined to be effective for ALA-PDT and / or ALA-PDD. And [10] phospho-ser15p53 protein is detected using an anti-phospho-ser15p53 antibody.
さらに本発明は、［１１］ＰＥＰＴ１の発現量を測定する試薬を備えたことを特徴とする、ＡＬＡ−ＰＤＴ又はＡＬＡ−ＰＤＤの施術前に５−アミノレブリン酸（ＡＬＡ）類を投与することの有効性を判定するためのキットや、［１２］ＰＥＰＴ１のｍＲＮＡを検出するためのプライマー対、若しくはプローブ、又はそれらの標識物を備えたことを特徴とする上記［１１］記載のキットや、［１３］ＰＥＰＴ１に特異的に結合する抗体、又は抗体の標識物を備えたことを特徴とする上記［１１］記載のキットや、［１４］がん抑制タンパク質ｐ５３の遺伝子変異を検出するための、がん抑制タンパク質ｐ５３の遺伝子の１１７〜１４２番目のコドン、１７１〜１８１番目のコドン、２３４〜２５８番目のコドン又は２７０〜２８６番目のコドンを含む１又は２以上の遺伝子をコードする塩基配列とストリンジェントな条件下でハイブリダイズするプローブを備えたことを特徴とする上記［１１］記載のキットや、［１５］さらに、ｐｈｏｓｐｈｏ−ｓｅｒ１５ｐ５３を検出するための、抗ｐｈｏｓｐｈｏ−ｓｅｒ１５ｐ５３抗体を備えたことを特徴とする上記［１１］〜［１４］のいずれか記載のキットに関する。 Furthermore, the present invention provides [11] a reagent for measuring the expression level of PEPT1, and is effective for administering 5-aminolevulinic acid (ALA) prior to ALA-PDT or ALA-PDD treatment. A kit for judging sex, [12] a kit according to [11] above, comprising a primer pair or probe for detecting PEPT1 mRNA, or a label thereof; [13] The kit according to [11] above, comprising an antibody that specifically binds to PEPT1, or a labeled product of the antibody, and [14] for detecting a gene mutation of the tumor suppressor protein p53. Codons 117-142, 171-181, 234-258, or 270-286 of the gene for tumor suppressor protein p53 Comprising a probe that hybridizes under stringent conditions with a nucleotide sequence encoding one or more genes including the above-mentioned [11], [15] and phospho-ser15p53 The kit according to any one of [11] to [14] above, which comprises an anti-phospho-ser15p53 antibody for detection.
ＡＬＡ−ＰＤＴ又はＡＬＡ−ＰＤＤの施術前に５−アミノレブリン酸類を投与することの有効性を予測することができる。 The effectiveness of administering 5-aminolevulinic acids prior to the treatment with ALA-PDT or ALA-PDD can be predicted.
本発明のＡＬＡ−ＰＤＴ又はＡＬＡ−ＰＤＤの施術前にＡＬＡ類を投与することの有効性を予測する方法としては、がん患者から採取した生体試料における、ＰＥＰＴ１（ペプチドトランスポーター１）の発現量の多寡を指標として、ＰＥＰＴ１の発現量が多いときに、ＡＬＡ−ＰＤＴ、及び／又は、ＡＬＡ−ＰＤＤに有効であると判定する有効性予測方法であれば特に制限されないが、がん抑制タンパク質ｐ５３の遺伝子変異の有無を検出し、遺伝子変異を検出しない場合、すなわち、がん抑制タンパク質ｐ５３の遺伝子が野生型である場合に、ＡＬＡ−ＰＤＴ、及び／又は、ＡＬＡ−ＰＤＤに有効であると判定する有効性予測方法を併用することが好ましい。 As a method for predicting the effectiveness of administering ALAs prior to the treatment of ALA-PDT or ALA-PDD of the present invention, the expression level of PEPT1 (peptide transporter 1) in a biological sample collected from a cancer patient As an index, there is no particular limitation as long as it is an efficacy prediction method that determines that it is effective for ALA-PDT and / or ALA-PDD when the expression level of PEPT1 is large. When the presence or absence of the gene mutation is detected and no gene mutation is detected, that is, when the gene of the tumor suppressor protein p53 is a wild type, it is determined to be effective for ALA-PDT and / or ALA-PDD It is preferable to use the effectiveness prediction method.
上記ＡＬＡ−ＰＤＴは、光に反応する化合物を投与し、光を照射することにより標的箇所を治療するＰＤＴを行う際に、それ自身は光増感作用を有さないＡＬＡ類を投与し、色素生合成経路を経て誘導されたＰｐＩＸががん細胞内に特異的に集積し、がん細胞内に蓄積したＰｐＩＸが、光照射されることにより周囲の酸素分子を光励起し、その結果生成する一重項酸素が、その強い酸化力による殺細胞効果を有することを利用するがんの治療方法であり、上記ＡＬＡ−ＰＤＤは、腫瘍の治療（手術）において、腫瘍の箇所を判断するＰＤＤを行う際に、それ自身は光増感作用を有さないＡＬＡ類を投与し、色素生合成経路を経て誘導されたＰｐＩＸががん細胞内に特異的に集積し、がん細胞内に蓄積したＰｐＩＸが青紫色の光（例えば４０５ｎｍ±１０ｎｍ）をあてると赤色の蛍光（例えば６３６ｎｍ±５ｎｍ）を発することを利用して、腫瘍の浸潤範囲を評価することにより、切除すべき腫瘍の箇所を判断する方法である。 The ALA-PDT administers a compound that reacts to light, and when performing PDT for treating a target site by irradiating light, administers ALAs that themselves do not have a photosensitizing action, PpIX induced through the biosynthetic pathway specifically accumulates in cancer cells, and PpIX accumulated in cancer cells photoexcites surrounding oxygen molecules when irradiated with light, resulting in a singlet It is a cancer treatment method utilizing the fact that oxygen of the term has a cell-killing effect due to its strong oxidizing power. In addition, ALAs that themselves do not have photosensitizing action are administered, and PpIX induced through the pigment biosynthesis pathway is specifically accumulated in cancer cells, and PpIX accumulated in cancer cells is Blue-violet light (for example, 405 m ± 10 nm) when exposed to by utilizing the fact that emits red fluorescence (e.g. 636 nm ± 5 nm), by evaluating the infiltration range of tumors, a method of determining the location of the tumor to be resected.
本発明の有効性予測方法に用いられるがん患者から採取した生体試料としては、がん患者の骨、脳組織、乳房組織、結腸組織、筋組織、神経組織、卵巣組織、前立腺組織、網膜組織、皮膚組織等のがん細胞や、脳脊髄液、心膜液、腹腔液、唾液、血清、尿等を含む体液の他、新生物を有することが疑われる任意の組織や、新生物を有すると考えられる任意の組織を挙げることができ、がん患者から採取する生体試料は標準的手順を用いて取り出すことができ、例えば、バイオプシーによって組織を取り出すことができる。 Examples of biological samples collected from cancer patients used in the efficacy prediction method of the present invention include bone, brain tissue, breast tissue, colon tissue, muscle tissue, nerve tissue, ovarian tissue, prostate tissue, and retinal tissue of cancer patients. In addition to cancer cells such as skin tissue and body fluids including cerebrospinal fluid, pericardial fluid, peritoneal fluid, saliva, serum, urine, etc., any tissue or neoplasm suspected of having a neoplasm is present. Any tissue that could be considered can be mentioned, and a biological sample taken from a cancer patient can be removed using standard procedures, for example, the tissue can be removed by biopsy.
本発明におけるＰＥＰＴ１の発現量を測定する方法としては、がん患者から採取した上記生体試料において、ＰＥＰＴ１遺伝子のｍＲＮＡを検出する方法や、ＰＥＰＴ１タンパク質の発現量を測定する方法を挙げることができる。 Examples of the method for measuring the expression level of PEPT1 in the present invention include a method for detecting mRNA of the PEPT1 gene in the biological sample collected from a cancer patient and a method for measuring the expression level of PEPT1 protein.
上記ＰＥＰＴ１遺伝子のｍＲＮＡを検出する方法としては、例えば、ＲＴ−ＰＣＲ法、ＦＩＳＨ法、ノーザンハイブリダイゼーション法、リアルタイムＰＣＲ法等を挙げることができる。これらの方法に用いられるプローブやプライマーは、ＰＥＰＴ１遺伝子の配列情報に基づいて適宜設計し、適当なオリゴヌクレオチド合成装置を用いて適宜作製することができる。 Examples of the method for detecting the mRNA of the PEPT1 gene include RT-PCR method, FISH method, Northern hybridization method, real-time PCR method and the like. Probes and primers used in these methods can be appropriately designed based on the sequence information of the PEPT1 gene, and can be appropriately produced using an appropriate oligonucleotide synthesizer.
上記ＲＴ−ＰＣＲ法としては、当業者において周知の技術を用いて容易に行なうことができるが、具体的には、被検者のがん細胞中のＰＥＰＴ１遺伝子の転写産物であるｍＲＮＡ又はその断片に対して相補的な一本鎖ＤＮＡを合成し、該ＤＮＡ又はその断片を特異的に増幅することができるプライマーにより該ＤＮＡ又はその断片をＰＣＲ増幅した後、増幅産物を電気泳動などで検出することができる。ＲＴ−ＰＣＲ法については、例えば、Ishikawaら, J.Clin. Oncol., 28:723-728, 1998などを参照して行なうこともできる。また、ＦＩＳＨ（Fluorescent in situ hybridization）法により、被検者のがん細胞中のＰＥＰＴ１遺伝子の転写状態を検出することができる。 The RT-PCR method can be easily performed using techniques well known to those skilled in the art. Specifically, mRNA or a fragment thereof, which is a transcript of the PEPT1 gene in cancer cells of a subject A single-stranded DNA complementary to the DNA is synthesized, the DNA or a fragment thereof is PCR-amplified with a primer capable of specifically amplifying the DNA or a fragment thereof, and the amplification product is detected by electrophoresis or the like be able to. The RT-PCR method can also be performed with reference to, for example, Ishikawa et al., J. Clin. Oncol., 28: 723-728, 1998. Moreover, the transcription | transfer state of PEPT1 gene in a subject's cancer cell is detectable by FISH (Fluorescent in situ hybridization) method.
上記ノーザンハイブリダイゼーション法におけるハイブリダイゼーションとしては、例えばモレキュラー・クローニング、ア・ラボラトリーマニュアル（Molecular cloning, A laboratory manual）、第３版、第９．５２−９．５５頁（１９８９）に記載の方法で行うことができる。使用されるプローブの標識化に用いられる標識物質としては、放射性同位元素、酵素、蛍光物質、発光物質、ランタニド元素、スピン試薬を例示することができる。上記放射性同位元素としては、〔１２５Ｉ〕、〔１３１Ｉ〕、〔３Ｈ〕、〔１４Ｃ〕、〔３２Ｐ〕、〔３３Ｐ〕、〔３５Ｓ〕、〔５９Ｆｅ〕を例示することができる。上記酵素としては、安定で比活性の大きなものが好ましく、β−ガラクトシダーゼ、β−グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素を例示することができ、上記蛍光物質としては、シアニン蛍光色素（Ｃｙ２、Ｃｙ３、Ｃｙ５、Ｃｙ５．５等）、フルオレスカミン、フルオレッセンイソチオシアネートを例示することができ、上記発光物質としては、ルミノール、ルミノール誘導体、ルシフェリンを例示することができる。 As the hybridization in the Northern hybridization method, for example, the method described in Molecular cloning, A laboratory manual, 3rd edition, pages 9.52-9.55 (1989) is used. It can be carried out. Examples of the labeling substance used for labeling the probe used include radioisotopes, enzymes, fluorescent substances, luminescent substances, lanthanide elements, and spin reagents. Examples of the radioisotopes include [ 125 I], [ 131 I], [ 3 H], [ 14 C], [ 32 P], [ 33 P], [ 35 S], [ 59 Fe]. Can do. The enzyme is preferably stable and has high specific activity, and examples thereof include β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, and the fluorescent substance includes cyanine fluorescent dye. (Cy2, Cy3, Cy5, Cy5.5, etc.), fluorescamine, fluorescein isothiocyanate can be exemplified, and examples of the luminescent substance include luminol, luminol derivatives, and luciferin.
上記リアルタイムＰＣＲ法としては、例えば、細胞内のトータルＲＮＡやｍＲＮＡから逆転写酵素を用いてｃＤＮＡを合成し、このｃＤＮＡを鋳型に目的領域をＰＣＲで増幅し、リアルタイムモニタリング用試薬を用いて増幅産物の生成過程をリアルタイムでモニタリングし解析する方法を挙げることができる。リアルタイムモニタリング試薬としては、例えば、ＳＹＢＲ（登録商標：Molecular Probes社製）ＧｒｅｅｎＩや、ＴａｑＭａｎ（登録商標：Applied Biosystems社製）プローブ等を挙げることができる。 As the real-time PCR method, for example, cDNA is synthesized from intracellular total RNA or mRNA using reverse transcriptase, the target region is amplified by PCR using this cDNA as a template, and amplified product using a reagent for real-time monitoring. A method of monitoring and analyzing the generation process of s in real time can be mentioned. Examples of the real-time monitoring reagent include SYBR (registered trademark: manufactured by Molecular Probes) Green I, TaqMan (registered trademark: manufactured by Applied Biosystems) probe, and the like.
前記ＰＥＰＴ１タンパク質の発現量を測定する方法としては、がん患者から採取した生体試料において、ウエスタンブロット法やＥＬＩＳＡ法を用いて測定する方法を例示することができる。 Examples of the method for measuring the expression level of the PEPT1 protein include a method of measuring a biological sample collected from a cancer patient using a Western blot method or an ELISA method.
上記ウエスタンブロット法としては、タンパク質を、ＳＤＳを含むポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）で分画し、ニトロセルロースフィルターに移し、抗体により検出する方法を挙げることができ、フィルターへ付着したタンパク質はこれらタンパク質に対する抗体をプローブとすることによって同定できる。抗体検出には標識化二次抗体法を用いることができる。ゲルからフィルターへのトランスファーは多くの場合エレクトロブロッティングによって行われる。ＰＥＰＴ１がコードするタンパク質に対する抗体はモノクローナル抗体のほかポリクローナル抗体でもよく、これらの抗体は常法により作製することができる。 Examples of the Western blotting method include a method in which a protein is fractionated by polyacrylamide gel electrophoresis (SDS-PAGE) containing SDS, transferred to a nitrocellulose filter, and detected by an antibody. Can be identified by using antibodies against these proteins as probes. A labeled secondary antibody method can be used for antibody detection. Gel to filter transfer is often performed by electroblotting. The antibody against the protein encoded by PEPT1 may be a monoclonal antibody or a polyclonal antibody, and these antibodies can be prepared by a conventional method.
上記ＥＬＩＳＡ法としては、サンドイッチ法（非競合法）と競合法の二つを挙げることができるが、サンドイッチ法によれば、マイクロプレートのウェルやプラスチックチューブなどの固相に、本発明におけるＰＥＰＴ１等の、目的物質に対する抗体をあらかじめ結合させておき、これにサンプルを添加してサンプル中の目的物質を抗原抗体反応により固相に結合させ、夾雑物を洗い流した後、酵素標識した第二の抗体を添加すると再度抗原抗体反応が起きることで、固相化抗体−目的物質−酵素標識抗体のサンドイッチ構造が構築され、その後遊離の酵素標識抗体を洗い流し、発色基質を添加することで、サンドイッチ構造の量（すなわちサンプル中の目的物質量）に比例して生起する発色反応により目的物質量を定量することができる。一方、競合法によれば、あらかじめ目的物質に対する抗体を結合させた固相に、サンプルと酵素標識抗原を添加して抗原抗体複合体を形成させ、固相に結合しなかった酵素標識抗原を洗い流した後に発色基質を添加し、生成した発色物質の吸光度を吸光度計で測定することによりサンプル中の目的物を定量することができる。 Examples of the ELISA method include a sandwich method (non-competitive method) and a competitive method. According to the sandwich method, PEPT1 or the like in the present invention is applied to a solid phase such as a well of a microplate or a plastic tube. The antibody to the target substance is bound in advance, the sample is added thereto, the target substance in the sample is bound to the solid phase by the antigen-antibody reaction, the contaminants are washed away, and then the enzyme-labeled second antibody As a result, an antigen-antibody reaction occurs again, and a sandwich structure of immobilized antibody-target substance-enzyme-labeled antibody is constructed. Then, the free enzyme-labeled antibody is washed away, and a chromogenic substrate is added. The amount of the target substance can be quantified by a color reaction that occurs in proportion to the amount (that is, the amount of the target substance in the sample). On the other hand, according to the competition method, an antigen-antibody complex is formed by adding a sample and an enzyme-labeled antigen to a solid phase in which an antibody against the target substance is bound beforehand, and the enzyme-labeled antigen that has not bound to the solid phase is washed away. After that, a chromogenic substrate is added, and the absorbance of the generated chromogenic substance is measured with an absorptiometer, whereby the target substance in the sample can be quantified.
本発明におけるｐ５３タンパク質遺伝子の変異を検出する方法としては、１塩基置換（点突然変異）等の遺伝子変異部位を検出することができる方法であれば特に制限されないが、かかる変異を野生型のがん抑制タンパク質ｐ５３の遺伝子の塩基配列と上記ｐ５３タンパク質遺伝子の塩基配列とを対比することにより見い出すために、ジデオキシヌクレオチドを用いてＤＮＡポリメラーゼの合成を塩基特異的に停止させるジデオキシ法等の、公知のダイレクトシークエンシング法を好適に例示することができる。また、特定位置の塩基置換だけを検出する場合に有用な、２０塩基の合成オリゴヌクレオチドと標的塩基配列とのハイブリッド形成の有無を利用したアレル特異的オリゴヌクレオチドハイブリダイゼーション法（ＭＡＳＡ法）（Hum. Mutation 2, 112-117, 1993）や、ＤＮＡ配列をＰＣＲで増幅する際に、その一端にＧＣに富んだ塩基配列（ＧＣクランプ）をつけ、変性剤濃度勾配ゲル電気泳動（denaturing gradient gel electrophoresis：ＤＧＧＥ）を行って１塩基置換を検出する方法（ＰＣＲ−ＤＧＧＥ法）（Proc. Natl. Acad. Sci. USA 86, 232-236, 1989）や、特定の遺伝子領域をＰＣＲ法により増幅して得られた増幅産物を試料ＤＮＡとして、２本鎖ＤＮＡ断片を１本鎖に解離し、熱変性させた一本鎖ＤＮＡが僅かな塩基配列の違いにより高次構造に変化することを応用したＰＣＲ−ＳＳＣＰ法（Single Strand Conformation Polymorphism：一本鎖コンフォメーション多型解析法）や、塩基配列特異的プローブ（ＳＳＯＰ）を用いたハイブリダイゼーション法（ＰＣＲ−ＳＳＯＰ法）や、ある領域をＰＣＲ法で増幅した後、点突然変異が生じた部位を切断部位と認識する制限酵素で処理して点突然変異を生じなかったＤＮＡとはサイズの違ったアリルとして検出するＰＣＲ−ＲＦＬＰ法（特開平５−３０８９９９号公報）や、クローン化された遺伝子やＤＮＡ断片を非アイソトープ化合物で標識後、スライドグラス上の染色体ＤＮＡとハイブリダイゼーションし、その分子雑種形成部位を蛍光シグナルとして直接染色体上に検出する方法であるＤＮＡ ＦＩＳＨ法（ＤＮＡ fluorescence in situ hybridization）等を用いることができる。 The method for detecting a mutation in the p53 protein gene in the present invention is not particularly limited as long as it is a method capable of detecting a gene mutation site such as a single base substitution (point mutation). In order to find out by comparing the base sequence of the gene of the tumor suppressor protein p53 and the base sequence of the p53 protein gene, a known method such as the dideoxy method for base-specifically stopping the synthesis of DNA polymerase using dideoxynucleotides is used. A direct sequencing method can be preferably exemplified. In addition, an allele-specific oligonucleotide hybridization method (MASA method) using the presence or absence of hybridization between a 20-base synthetic oligonucleotide and a target base sequence, which is useful when detecting only a base substitution at a specific position (Hum. Mutation 2, 112-117, 1993) or when a DNA sequence is amplified by PCR, a GC-rich base sequence (GC clamp) is attached to one end of the DNA sequence, and denaturing gradient gel electrophoresis: DGGE) to detect single base substitution (PCR-DGGE method) (Proc. Natl. Acad. Sci. USA 86, 232-236, 1989), or a specific gene region obtained by PCR amplification Using the amplified product as a sample DNA, dissociating double-stranded DNA fragments into single strands, and applying heat-denatured single-stranded DNA to higher-order structures due to slight differences in base sequences PCR-SSCP method (Single Strand Conformation Polymorphism), hybridization method using base sequence specific probe (SSOP) (PCR-SSOP method), PCR method for certain regions PCR-RFLP method in which a site where a point mutation has occurred is treated with a restriction enzyme that recognizes it as a cleavage site and then detected as an allele having a different size from that of the DNA that did not cause a point mutation. No. 308999) or a method of detecting a cloned gene or DNA fragment with a non-isotope compound and then hybridizing with a chromosomal DNA on a slide glass and directly detecting its molecular hybrid formation site on a chromosome as a fluorescent signal DNA FISH method (DNA fluorescence in situ hybridization) etc. can be used
かかる塩基配列の決定により見い出される本発明におけるがん抑制タンパク質ｐ５３としては、ＧｅｎＢａｎｋアクセッション番号ＮＰ＿０００５３７で表されるアミノ酸配列を有するタンパク質を例示することができ、がん抑制タンパク質ｐ５３の遺伝子（例えば、ＧｅｎＢａｎｋアクセッション番号ＮＭ＿０００５４６で表される塩基配列を有する遺伝子）の変異としては、野生型の（正常な）がん抑制タンパク質ｐ５３の遺伝子のＤＮＡの塩基配列において、少なくとも１の塩基の変異の結果、アミノ酸配列レベルで野生型のがん抑制タンパク質ｐ５３をコードする遺伝子とはいえなくなる遺伝子変異を挙げることができ、ここにおける塩基の変異の種類としては、当該塩基の他の塩基への置換や、当該塩基の欠失や、当該塩基の配列部位への他の塩基の挿入を例示することができる。かかる遺伝子変異の存在する位置としては、がん抑制タンパク質ｐ５３の遺伝子の中央部のＤＮＡ結合ドメインを含むコアドメイン（１０２〜２９２番目のコドン）における、ホットスポットと呼ばれる変異頻度のきわめて高い４ヶ所の保存領域であるII;１１７〜１４２番目のコドン、III;１７１〜１８１番目のコドン、IV;アミノ酸コドン２３４〜２５８番目のコドン、V;２７０〜２８６番目のコドンを例示することができ、具体的には、２４５番目のコドンにおいて見い出される、塩基配列ＧＧＣがＧＡＣに変異することによるグリシン（Ｇｌｙ）からアスパラギン酸（Ａｓｐ）へのアミノ酸の変異や、２７３番目のコドンにおいて見い出される、塩基配列ＣＧＴがＣＡＴに変異することによるアルギニン（Ａｒｇ）からヒスチジン（Ｈｉｓ）へのアミノ酸の変異（Proc. Natl. Acad Sci. USA. 87 : 7555 -7559, 1990）や、１７３番目のコドンにおいて見い出される、バリン（Ｖａｌ）からメチオニン（Ｍｅｔ）へのアミノ酸の変異(Gut. 44:366-71, 1999)等を挙げることができる。 As the tumor suppressor protein p53 in the present invention found by the determination of such a base sequence, a protein having the amino acid sequence represented by GenBank accession number NP_000537 can be exemplified, and a gene of the tumor suppressor protein p53 (for example, As a mutation of the gene having the base sequence represented by GenBank accession number NM_000546), as a result of mutation of at least one base in the DNA base sequence of the wild-type (normal) tumor suppressor protein p53 gene, Examples of the gene mutation that can not be said to be a gene encoding the wild type tumor suppressor protein p53 at the amino acid sequence level include the substitution of the base with another base, Deletion of base and sequence of the base It can be exemplified insertion of another base to position. Such gene mutations are present at four sites with extremely high mutation frequency called hot spots in the core domain (codons 102 to 292) including the DNA binding domain in the center of the gene of the tumor suppressor protein p53. Examples of the conserved region II include: codons 117 to 142, III; codons 171 to 181; IV; amino acid codons 234 to 258, and V; 270 to 286. The amino acid mutation from glycine (Gly) to aspartic acid (Asp) found by mutating the base sequence GGC to GAC found in the 245th codon, or the base sequence CGT found in the 273th codon Arginine (Arg) to histidine (His) by mutating to CAT Amino acid mutation (Proc. Natl. Acad Sci. USA. 87: 7555 -7559, 1990) and the amino acid mutation from valine (Val) to methionine (Met) found at the 173rd codon (Gut. 44: 366-71, 1999).
本発明の有効性予測方法において、がん患者から採取した生体試料において、ＡＬＡ−ＰＤＴ、及び／又は、ＡＬＡ−ＰＤＤ施術前に、１５番目のセリンの位置でリン酸化しているがん抑制タンパク質ｐ５３を検出することで、ＡＬＡがＡＬＡ−ＰＤＴに有効であると判断する方法を併用することが好ましい。ｐ５３はＤＮＡ損傷、ＵＶ照射、化学療法剤や放射線処理などによるストレス応答によって活性化を受け、細胞をアポトーシスに導く。活性化される前のｐ５３タンパクの１８〜２６番目の領域にはユビキチンリガーゼのＭＤＭ２タンパクが結合し、ユビキチン−プロテアソーム系によるｐ５３の分解が促進され、不活性化されている。ストレスに応答した細胞ではＡＴＭ(放射線によるＤＮＡの二本鎖切断に反応)やＡＴＲ（紫外線と反応)等のリン酸化酵素が活性化し、ｐ５３の１５番目のセリンがリン酸化される。この１５番目のセリンのリン酸化はｐ５３の安定化や転写活性を亢進させると考えられている。１５番目のセリンの位置でリン酸化しているがん抑制タンパク質ｐ５３の検出方法としては、前記ウエスタンブロット法やＥＬＩＳＡ法をあげることができる。 In the effectiveness prediction method of the present invention, in a biological sample collected from a cancer patient, a tumor suppressor protein phosphorylated at the 15th serine position before ALA-PDT and / or ALA-PDD treatment It is preferable to use together a method for determining that ALA is effective for ALA-PDT by detecting p53. p53 is activated by a stress response caused by DNA damage, UV irradiation, chemotherapeutic agents or radiation treatment, and leads to apoptosis of cells. The MDM2 protein of ubiquitin ligase binds to the 18th to 26th region of the p53 protein before being activated, and the degradation of p53 by the ubiquitin-proteasome system is promoted and inactivated. In cells responding to stress, phosphorylating enzymes such as ATM (reacting to double-strand breakage of DNA by radiation) and ATR (reacting with ultraviolet light) are activated, and the 15th serine of p53 is phosphorylated. This 15th serine phosphorylation is believed to enhance p53 stabilization and transcriptional activity. Examples of the method for detecting the tumor suppressor protein p53 phosphorylated at the 15th serine position include the Western blot method and the ELISA method.
上記ＡＬＡ−ＰＤＴ又はＡＬＡ−ＰＤＤにおいて投与されるＡＬＡ類としては、ＡＬＡ若しくはその誘導体又はこれらの塩が含まれ、δ−アミノレブリン酸とも呼ばれるＡＬＡは、式ＨＯＯＣ−（ＣＨ2)2−（ＣＯ）−ＣＨ2−ＮＨ2で表されるアミノ酸の一種であり、例えば特開平４−９３６０号公報等に記載された化学合成法や、微生物による生産、酵素による生産等のいずれの公知の方法によっても製造することができ、粗精製物は、免疫機能向上に対して有害な物質を含まない限り分離精製することなくそのまま用いることができる。 ALAs administered in the ALA-PDT or ALA-PDD include ALA or a derivative thereof or a salt thereof, and ALA also called δ-aminolevulinic acid is represented by the formula HOOC- (CH 2 ) 2- (CO). It is a kind of amino acid represented by —CH 2 —NH 2 , and can be obtained by any known method such as a chemical synthesis method described in JP-A-4-9360, production by microorganisms, production by enzymes, etc. The crudely purified product can be used as it is without being separated and purified as long as it does not contain substances harmful to immune function improvement.
上記ＡＬＡ類のうち、ＡＬＡの誘導体としては、ＡＬＡのエステル基とアシル基を有するものを挙げることができ、好ましくはメチルエステル基とホルミル基、メチルエステル基とアセチル基、メチルエステル基とｎ−プロパノイル基、メチルエステル基とｎ−ブタノイル基、エチルエステル基とホルミル基、エチルエステル基とアセチル基、エチルエステル基とｎ−プロパノイル基、エチルエステル基とｎ−ブタノイル基の組合せを例示することができる。 Among the ALAs, examples of ALA derivatives include those having an ALA ester group and an acyl group, preferably a methyl ester group and a formyl group, a methyl ester group and an acetyl group, and a methyl ester group and an n- group. Examples of combinations of propanoyl group, methyl ester group and n-butanoyl group, ethyl ester group and formyl group, ethyl ester group and acetyl group, ethyl ester group and n-propanoyl group, ethyl ester group and n-butanoyl group it can.
また、ＡＬＡ類のうち、ＡＬＡ又はその誘導体の塩としては、例えば塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、リン酸塩、硝酸塩、硫酸塩、酢酸塩、プロピオン酸塩、トルエンスルホン酸塩、コハク酸塩、シュウ酸塩、乳酸塩、酒石酸塩、グリコール酸塩、メタンスルホン酸塩、酪酸塩、吉草酸塩、クエン酸塩、フマル酸塩、マレイン酸塩、リンゴ酸塩等の酸付加塩、及びナトリウム塩、カリウム塩、カルシウム塩等の金属塩、アンモニウム塩、アルキルアンモニウム塩等を挙げることができる。なお、これらの塩は使用時において溶液として用いられ、その作用はＡＬＡ及びその誘導体の場合と同一である。以上のＡＬＡ類は、水和物又は溶媒和物を形成していてもよく、いずれかを単独で又は２種類以上を適宜組み合わせて用いることができる。 Among the ALAs, examples of salts of ALA or its derivatives include, for example, hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfone Acid salt, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate etc Examples thereof include acid addition salts, metal salts such as sodium salts, potassium salts, and calcium salts, ammonium salts, and alkylammonium salts. These salts are used as a solution at the time of use, and the action is the same as in the case of ALA and its derivatives. The above ALAs may form hydrates or solvates, and can be used alone or in combination of two or more.
本発明のＡＬＡ類の有効性を判定するためのキットとしては、ＰＥＰＴ１の発現量を測定する試薬を備えたＡＬＡ−ＰＤＴ又はＡＬＡ−ＰＤＤの施術前にＡＬＡ類を投与することの有効性を判定するためのキットであれば特に制限されず、例えば、ＰＥＰＴ１遺伝子のｍＲＮＡを検出するためのプライマー対、若しくはプローブ、又はそれらの標識物を備えたキットや、ＰＥＰＴ１の発現量を測定するためのＰＥＰＴ１に特異的に結合する抗体、抗体の標識物等を備えたキットや、がん抑制タンパク質ｐ５３の遺伝子の変異を検出するための、がん抑制タンパク質ｐ５３の遺伝子の１１７〜１４２番目のコドン、１７１〜１８１番目のコドン、２３４〜２５８番目のコドン、２７０〜２８６番目のコドン等に位置する１又は２以上の遺伝子をコードする塩基配列とストリンジェントな条件下でハイブリダイズするプローブを備えたキットを挙げることができ、さらに、ｐｈｏｓｐｈｏ−ｓｅｒ１５ｐ５３タンパク質を検出するための、抗ｐｈｏｓｐｈｏ−ｓｅｒ１５ｐ５３抗体を備えるキットが好ましく、これらのキットを用いることにより、がん治療における５−アミノレブリン酸（ＡＬＡ）の有効性をＡＬＡ−ＰＤＴ、及び／又は、ＡＬＡ−ＰＤＤの施術前により有効に予測することができる。 As a kit for determining the effectiveness of ALAs of the present invention, the effectiveness of administering ALAs prior to the treatment of ALA-PDT or ALA-PDD equipped with a reagent for measuring the expression level of PEPT1 is determined. For example, a kit provided with a primer pair or probe for detecting the mRNA of the PEPT1 gene, or a label thereof, or PEPT1 for measuring the expression level of PEPT1 A kit having an antibody that specifically binds to the antibody, a label of the antibody, and the like, a codon at positions 117 to 142 of the gene of the tumor suppressor protein p53 for detecting a mutation in the gene of the tumor suppressor protein p53, 171 One or more genes located at the 181st codon, the 234th to 258th codon, the 270th to 286th codon, etc. And a kit having a probe that hybridizes under stringent conditions with a base sequence to be loaded, and a kit having an anti-phospho-ser15p53 antibody for detecting a phospho-ser15p53 protein is preferred. By using this kit, the effectiveness of 5-aminolevulinic acid (ALA) in cancer treatment can be predicted more effectively before ALA-PDT and / or ALA-PDD treatment.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
（細胞の種類によるＡＬＡ−ＰＤＴ効果の違い） (Difference in ALA-PDT effect depending on cell type)
ヒト胃がん細胞株ＭＫＮ−４５、ヒト胃がん細胞株ＫＫＬＳ、ヒト胃がん細胞株ＴＭＫ−１、及びヒト大腸がん細胞株ＨＴ−２９の各細胞株を３５ｍｍディッシュに２×１０５個播種し、１６時間後ＡＬＡ塩酸塩（コスモバイオ社製）最終濃度２００μＭを添加し、６時間後にＤＬＤ−Ｒ２レーザー照射装置（ミワテック）を用いて、２００ｍＷ、５ｍｉｎ、２１．６Ｊ／ｃｍ２相当を照射してＡＬＡ−ＰＤＴを行った。照射２４時間後、培地を除去し、ＰＢＳ溶液にクリスタルバイオレット溶液（0.5% crystal violet in 25% methanol）を添加し、室温にて５分反応させた。Crystal violet溶液を捨て、流水で洗い、２％デオキシコール酸ナトリウム溶液に溶解させ、室温で１０分放置した。イムノリーダー（日本インターメッド社製）を用いて６２０ｎｍにおける吸光度を測定し、以下の式を用いて細胞増殖抑制率（inhibition ratio: IR）を算出した。
細胞増殖抑制率（％）＝（１−Ｔ／Ｃ）×１００
（但し、Ｔは各濃度における処理群の平均吸光度、Ｃは対照群の平均吸光度）
結果を以下の表１に示す。
Human gastric cancer cell line MKN-45, human gastric cancer cell line KKLS, seeded 2 × 10 5 cells in human gastric cancer cell line TMK-1, and each cell line of human colon cancer cell line HT-29 35 mm dish, 16 hours After adding ALA hydrochloride (manufactured by Cosmo Bio) at a final concentration of 200 μM, 6 hours later, using a DLD-R2 laser irradiation device (Miwatec), irradiation with an equivalent of 200 mW, 5 min, 21.6 J / cm 2 and ALA- PDT was performed. After 24 hours of irradiation, the medium was removed, and a crystal violet solution (0.5% crystal violet in 25% methanol) was added to the PBS solution, followed by reaction at room temperature for 5 minutes. The Crystal violet solution was discarded, washed with running water, dissolved in 2% sodium deoxycholate solution, and allowed to stand at room temperature for 10 minutes. The absorbance at 620 nm was measured using an immunoleader (manufactured by Nihon Intermed), and the cell growth inhibition ratio (IR) was calculated using the following formula.
Cell growth inhibition rate (%) = (1−T / C) × 100
(Where T is the average absorbance of the treatment group at each concentration, and C is the average absorbance of the control group)
The results are shown in Table 1 below.
次に、各薬剤濃度におけるＩＲ値を片対数グラフにプロットし、容量反応曲線よりＩＣ５０値を求めた。その結果を表２に示す。各細胞腫でＩＣ５０値に差が認められた。特にＭＫＮ−４５では、低濃度でＩＲが高かった。 Next, the IR value at each drug concentration was plotted on a semilogarithmic graph, and the IC 50 value was determined from the volume response curve. The results are shown in Table 2. There was a difference in IC 50 values for each cell tumor. In particular, with MKN-45, IR was high at low concentrations.
［結果］
ＡＬＡ−ＰＤＴ最終濃度２００μＭを添加した場合、ＡＬＡ−ＰＤＴの効果は、ヒト胃がん細胞株ＭＫＮ−４５において最も大きく、ヒト胃がん細胞株ＴＭＫ−１においては最も小さいことを確認した。
[result]
When ALA-PDT final concentration of 200 μM was added, it was confirmed that the effect of ALA-PDT was the greatest in human gastric cancer cell line MKN-45 and the smallest in human gastric cancer cell line TMK-1.
（ＰＥＰＴ１遺伝子のｍＲＮＡの検出）
実施例１で用いた細胞株から全ｍＲＮＡを採取し、ヒトＰＥＰＴ１遺伝子[GenBank:SLC15A1 NM_005073]又はヒトＰＥＰＴ２遺伝子[GenBank:SLC15A2 NM_021082]中の特定の配列を増幅するために、以下のオリゴヌクレオチドプライマーを用いてＲＴ−ＰＣＲを実施し、ＰＥＰＴ１遺伝子又はＰＥＰＴ２遺伝子の断片を増幅した。
ＰＥＰＴ１遺伝子に対するプライマー：5’-GAGGGCCAGGAGAAACAAAGAAACAG-3’(forward)(配列番号１)及び5’-CAGGAACATCACCCTCGTAACCATCT-3’(reverse)(配列番号２)
（増幅産物４０２ｂｐ）。
ＰＥＰＴ２遺伝子に対するプライマー：
5’-AGCCATGAATCCTTTCCAGAAAAATGAGTC-3’(forward)(配列番号３)及び5’-AATCAAGCTCCCTGCATTGATGGAC-3’(reverse)(配列番号４)
（増幅産物５９５ｂｐ）。
(Detection of mRNA of PEPT1 gene)
In order to amplify a specific sequence in the human PEPT1 gene [GenBank: SLC15A1 NM_005073] or the human PEPT2 gene [GenBank: SLC15A2 NM_021082] from the cell line used in Example 1, the following oligonucleotide primers were used. RT-PCR was carried out using the above to amplify the PEPT1 gene or PEPT2 gene fragment.
Primer for PEPT1 gene: 5'-GAGGGCCAGGAGAAACAAAGAAACAG-3 '(forward) (SEQ ID NO: 1) and 5'-CAGGAACATCACCCTCGTAACCATCT-3' (reverse) (SEQ ID NO: 2)
(Amplification product 402 bp).
Primer for PEPT2 gene:
5'-AGCCATGAATCCTTTCCAGAAAAATGAGTC-3 '(forward) (SEQ ID NO: 3) and 5'-AATCAAGCTCCCTGCATTGATGGAC-3' (reverse) (SEQ ID NO: 4)
(Amplification product 595 bp).
さらに、全ｍＲＮＡ量について、以下のオリゴヌクレオチドプライマーを用いて各組織におけるβ−アクチンレベルを測定することにより標準化した。
β−アクチン遺伝子[GenBank:X00351]に対するプライマー：5’-GAAAATCTGGCACCACACCTT-3’(forward)(配列番号５)及び5’-TTGAAGGTAGTTTCGTGGAT-3’(reverse)(配列番号６)
（増幅産物５９１ｂｐ）。
結果を図１に示す。
Furthermore, the total mRNA amount was normalized by measuring the β-actin level in each tissue using the following oligonucleotide primers.
Primers for β-actin gene [GenBank: X00351]: 5′-GAAAATCTGGCACCACACCTT-3 ′ (forward) (SEQ ID NO: 5) and 5′-TTGAAGGTAGTTTCGTGGAT-3 ′ (reverse) (SEQ ID NO: 6)
(Amplification product 591 bp).
The results are shown in FIG.
［結果］
実施例１でＡＬＡ−ＰＤＴで用いた細胞株のうち、ＩＣ５０値の低い細胞ほど、ＰＥＰＴ１遺伝子のｍＲＮＡ発現量が高かったことが示された（図１参照）。このことは、ＰＥＰＴ１の発現が高いほどＡＬＡの取込みが多く、ＡＬＡ−ＰＤＴの効果が大きいものと示唆された。
[result]
Among the cell lines used in ALA-PDT in Example 1, it was shown that the lower the IC 50 value, the higher the mRNA expression level of the PEPT1 gene (see FIG. 1). This suggested that the higher the expression of PEPT1, the more ALA uptake and the greater the effect of ALA-PDT.
ＰＥＰＴ１遺伝子のｍＲＮＡ発現とＡＬＡ取込み量との関連性を明らかにするため、実施例１で用いた細胞株において、ＡＬＡを添加後の細胞内におけるＰｐＩＸ量をＨＰＬＣ法により測定し、ＰＥＰＴ１遺伝子のｍＲＮＡ発現量と比較した。各種ヒトがん細胞１ｘ１０７ｃｅｌｌｓを１５ｃｍディッシュに播種し、２４時間後２００μＭのＡＬＡを培地に添加した。培養４時間後に細胞を回収し、ＨＰＬＣにより細胞内ＰｐＩＸを測定し、ＰｐＩＸを算出した。結果を以下の表３に示す。 In order to clarify the relationship between the mRNA expression of PEPT1 gene and the amount of ALA uptake, in the cell line used in Example 1, the amount of PpIX in the cells after addition of ALA was measured by HPLC method, and the mRNA of PEPT1 gene The expression level was compared. Various human cancer cells 1 × 10 7 cells were seeded in a 15 cm dish, and after 24 hours, 200 μM ALA was added to the medium. After 4 hours of culture, the cells were collected, intracellular PpIX was measured by HPLC, and PpIX was calculated. The results are shown in Table 3 below.
［結果］
ＰＥＰＴ１の発現を認めたＭＫＮ−４５において高濃度のＰｐＩＸが検出され、ＡＬＡ取込み量はＰＥＰＴ１の発現量に相関するものと示唆された。
[result]
A high concentration of PpIX was detected in MKN-45 in which the expression of PEPT1 was observed, suggesting that the ALA uptake correlated with the expression level of PEPT1.
（各細胞における変異ｐ５３変異）
がん細胞におけるｐ５３変異の解析では、まずアミノ酸コード領域内のホットスポットを含むｃＤＮＡ（GenBank:NM_000546）断片をＲＴ−ＰＣＲにより作製し、ダイレクトシークエンシング法によりその塩基配列を決定した。ＤＮＡの塩基配列は、Thermo Sequenase Cy5.5 Dye Terminator Cycle Sequencing Kit（Amersham Biosciences社製）を用いて解析した。プラスミドＤＮＡ２μｇにreaction buffer３．５μＬ、２μＭプライマー２μＬ、Thermo Sequenase ＤＮＡポリメラーゼ２μＬを添加し、蒸留水で全量を３１．５μＬに調製し、反応混合溶液とした。予め１μＬずつ分注しておいたＤＮＡ／ｄｄＡＴＰ−ｍｉｘ、ＤＮＡ／ｄｄＣＴＰ−ｍｉｘ、ＤＮＡ／ｄｄＧＴＰ−ｍｉｘ、及びＤＮＡ／ｄｄＴＴＰ−ｍｉｘに反応混合溶液を７μＬずつ添加した。ＰＣＲ反応は、９４℃にて３分間の加熱後、９４℃にて５０秒、５７℃にて５０秒、７２℃にて２分を３２回繰り返した。反応終了後、各反応チューブに１０ｍｇ／ｍＬグリコーゲン溶液２μＬ、７．５Ｍ ammonium acetate ２μＬ、氷冷エタノール３０μＬを添加し、よく撹拌し、氷中に２０分間インキュベートした。４℃で１２，０００ｒｐｍ、３０分間遠心した後、上清を捨てた。２００μＬの７０％氷冷エタノールを添加し、４℃、１２，０００ｒｐｍ、５分間遠心し上清を捨てた後、乾燥させた。その後、６μＬのloading bufferを添加し、７０℃にて３分間の熱変性を行った。ＳＥＱ４×４ personal sequencing system（Amersham Biosciences社製）を用いて１,５００Ｖにて、４５分間の泳動後、ＳＥＱ４×４ Basecaller software（Amersham Biosciences）を用いてＤＮＡ塩基配列の解析を行った。ホットスポットを含む領域のＤＮＡ断片の作製に用いたプライマーは、
p53-37seq
(5’-ttgccgtcccaagcaatggatgatttgatgctgtc-3’)(forward)(配列番号７)
及び、
p53-2seq
(5’-gcggagattctcttcctctgtgcgccggtctctccca-3’)(reverse)(配列番号８)であった。
また、塩基配列の決定には以下のプライマーを用いた；
p53-37seq
(5’-ttgccgtcccaagcaatggatgatttgatgctgtc-3’)(forward)(配列番号９)、
p53-seq5
(5’-GATGAAGCTCCCAGAATGCCAGAGGCTGCT-3’)(forward)(配列番号１０)、
p53-1seq
(5’-acctgccctgtgcagctgtgggttgattccaca-3’)(forward)(配列番号１１)、
p53-seq4
(5’-TTGCGTGTGGAGTATTTGGATGACAGAAACACTTTT-3’)(forward)(配列番号１２)、
p53-44seq
(5’-gcaagtcacagacttggctgtcccagaatgcaag-3’)(reverse)(配列番号１３)、
p53-seq6
(5’-tctcccaggacaggcacaaacatgca-3’)(reverse)(配列番号１４)、
p53-2seq
(5’-gcggagattctcttcctctgtgcgccggtctctccca-3’)(reverse)(配列番号１５)。
図２に結果を示す。
(Mutation p53 mutation in each cell)
In the analysis of p53 mutation in cancer cells, a cDNA (GenBank: NM_000546) fragment containing a hot spot in the amino acid coding region was first prepared by RT-PCR, and its nucleotide sequence was determined by direct sequencing. The base sequence of the DNA was analyzed using Thermo Sequenase Cy5.5 Dye Terminator Cycle Sequencing Kit (Amersham Biosciences). Reaction buffer 3.5 μL, 2 μM primer 2 μL, and Thermo Sequenase DNA polymerase 2 μL were added to 2 μg of plasmid DNA, and the total volume was adjusted to 31.5 μL with distilled water to obtain a reaction mixed solution. 7 μL of the reaction mixture solution was added to DNA / ddATP-mix, DNA / ddCTP-mix, DNA / ddGTP-mix, and DNA / ddTTP-mix that had been dispensed in an amount of 1 μL in advance. In the PCR reaction, after heating at 94 ° C for 3 minutes, 94 ° C for 50 seconds, 57 ° C for 50 seconds, and 72 ° C for 2 minutes were repeated 32 times. After completion of the reaction, 2 μL of 10 mg / mL glycogen solution, 2 μL of 7.5 M ammonium acetate, and 30 μL of ice-cold ethanol were added to each reaction tube, stirred well, and incubated in ice for 20 minutes. After centrifugation at 12,000 rpm for 30 minutes at 4 ° C., the supernatant was discarded. 200 μL of 70% ice-cold ethanol was added, centrifuged at 4 ° C., 12,000 rpm for 5 minutes, the supernatant was discarded, and then dried. Thereafter, 6 μL of loading buffer was added, and heat denaturation was performed at 70 ° C. for 3 minutes. After running for 45 minutes at 1,500 V using a SEQ4 × 4 personal sequencing system (manufactured by Amersham Biosciences), the DNA base sequence was analyzed using SEQ4 × 4 Basecaller software (Amersham Biosciences). Primers used for the preparation of the DNA fragment of the region containing the hot spot are:
p53-37seq
(5'-ttgccgtcccaagcaatggatgatttgatgctgtc-3 ') (forward) (SEQ ID NO: 7)
as well as,
p53-2seq
(5'-gcggagattctcttcctctgtgcgccggtctctccca-3 ') (reverse) (SEQ ID NO: 8).
In addition, the following primers were used to determine the base sequence;
p53-37seq
(5'-ttgccgtcccaagcaatggatgatttgatgctgtc-3 ') (forward) (SEQ ID NO: 9),
p53-seq5
(5'-GATGAAGCTCCCAGAATGCCAGAGGCTGCT-3 ') (forward) (SEQ ID NO: 10),
p53-1seq
(5'-acctgccctgtgcagctgtgggttgattccaca-3 ') (forward) (SEQ ID NO: 11),
p53-seq4
(5'-TTGCGTGTGGAGTATTTGGATGACAGAAACACTTTT-3 ') (forward) (SEQ ID NO: 12),
p53-44seq
(5'-gcaagtcacagacttggctgtcccagaatgcaag-3 ') (reverse) (SEQ ID NO: 13),
p53-seq6
(5'-tctcccaggacaggcacaaacatgca-3 ') (reverse) (SEQ ID NO: 14),
p53-2seq
(5'-gcggagattctcttcctctgtgcgccggtctctccca-3 ') (reverse) (SEQ ID NO: 15).
The results are shown in FIG.
［結果］
図２に示すとおり、上述のダイレクトシークエンシング法により、胃がん細胞株ＫＫＬＳでは２４５番目のアミノ酸がＧｌｙからＡｓｐに変異していることを確認した。また、大腸がん細胞株ＨＴ−２９では２７３番目のＡｒｇがＨｉｓに変異していることを確認したが、ヒト胃がん細胞株ＭＫＮ−４５は野性型のｐ５３遺伝子を有し、変異は確認されなかった。この結果は、ヒト胃がんＭＫＮ−４５株は野性型のｐ５３遺伝子を有し（Mol Carcinog. 19:243-53, 1997）、大腸がん細胞株ＨＴ−２９では２７３番目のＡｒｇがＨｉｓに変異しているという報告(Proc. Natl. Acad Sci. USA. 87 : 7555 -7559, 1990)と一致する。
[result]
As shown in FIG. 2, it was confirmed by the above-described direct sequencing method that the amino acid at position 245 was mutated from Gly to Asp in the gastric cancer cell line KKLS. Moreover, in the colon cancer cell line HT-29, it was confirmed that the 273rd Arg was mutated to His, but the human gastric cancer cell line MKN-45 has a wild type p53 gene, and no mutation was confirmed. It was. As a result, human gastric cancer strain MKN-45 has a wild type p53 gene (Mol Carcinog. 19: 243-53, 1997). In colorectal cancer cell line HT-29, the 273rd Arg is mutated to His. (Proc. Natl. Acad Sci. USA. 87: 7555-7559, 1990).
（ＡＬＡ−ＰＤＴにより誘導されるｐ５３の安定性とｓｅｒ１５のリン酸化に対するがん抑制タンパク質ｐ５３特異的阻害剤の影響）
３５ｍｍ培養シャーレ上にＭＫＮ−４５(１ｘ１０６ｃｅｌｌｓ)を播種し、２４時間３７℃にて、５％ＣＯ２インキュベーター内で培養した。ＡＬＡ塩酸塩を最終濃度０μＭ、５０μＭ、１００μＭ、２００μＭになるように添加し、さらにｐｉｆｉｔｈｒｉｎ−αを、最終濃度５０μＭになるようにＡＬＡ塩酸塩と同時に添加した。薬剤処理後４時間から６時間培養を行い、ＤＬＤ−Ｒ２レーザー照射装置を用いて、２００ｍＷ、４又は２分間、５又は２．５Ｊ／ｃｍ２相当を照射して、ＡＬＡ−ＰＤＴを行った。２４時間後、死細胞を含む培養上清を１５ｍＬのコニカルチューブに回収し、ＰＢＳ（−）溶液により洗浄した。付着細胞はＲＩＰＡ ｂｕｆｆｅｒ[150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 0.1% SDS, 1% デオキシコール酸ナトリウム, 1％ NP-40 (ナカライテスク株式会社製)、1mM フェニルメチルスルフォニルフルオライド（ＰＭＳＦ）、0.5％プロテアーゼインヒビターカクテル(SIGMA社製)]を用いて溶解し、先に回収した浮遊細胞ペレットに添加し、混合した。細胞溶解液は４℃にて３０分間振盪し、１５,０００ｒｐｍ、１０分間の遠心により不溶物を除去し、ウエスタンブロット解析に用いた。
(Influence of p53-specific inhibitor of tumor suppressor protein on the stability of p53 and ser15 phosphorylation induced by ALA-PDT)
MKN-45 (1 × 10 6 cells) was seeded on a 35 mm culture dish, and cultured for 24 hours at 37 ° C. in a 5% CO 2 incubator. ALA hydrochloride was added to final concentrations of 0 μM, 50 μM, 100 μM, and 200 μM, and pifithrin-α was added simultaneously with ALA hydrochloride to a final concentration of 50 μM. Cultivation was performed for 4 to 6 hours after drug treatment, and ALA-PDT was performed using a DLD-R2 laser irradiator and irradiated with 200 mW, 4 or 2 minutes, 5 or 2.5 J / cm 2 equivalent. After 24 hours, the culture supernatant containing dead cells was collected in a 15 mL conical tube and washed with a PBS (−) solution. Adherent cells were RIPA buffer [150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 0.1% SDS, 1% sodium deoxycholate, 1% NP-40 (manufactured by Nacalai Tesque, Inc.), 1 mM phenylmethylsulfonyl fluoride. (PMSF), 0.5% protease inhibitor cocktail (manufactured by SIGMA)], and added to the previously collected floating cell pellet and mixed. The cell lysate was shaken at 4 ° C. for 30 minutes, insolubles were removed by centrifugation at 15,000 rpm for 10 minutes, and used for Western blot analysis.
タンパク抽出液に試料溶解液（50 mM Tris-HCl (pH 6.5), 10% glycerol, 2% SDS, 0.1% bromophenol blue, 40 mM dithiothreitol (DTT)）を添加後、９４℃にて３分間の熱変性を行い、泳動緩衝溶液（20 mM Tris, 200 mM glycine, 0.3% SDS）中、３６ｍＡの定電流で１２．５％ ｅ−ＰＡＧＥＬ(ATTO株式会社製)を用いて電気泳動を行った。泳動後のタンパクをポリビニリデンフルオライド（ＰＶＤＦ）膜（日本ミリポア・リミテッド社製）に転写した後、２．５％のスキムミルクを溶解した５％Ｔｗｅｅｎ−２０添加ＴＢＳ(TBS-Tween)溶液中室温で３０分間ＰＶＤＦ膜を振盪させた。その後、０．２５％スキムミルクを含むＴＢＳ−Ｔｗｅｅｎ溶液(洗浄液)で１０分、５分、５分と３回ＰＶＤＦ膜を洗浄した。５％Ｔｗｅｅｎ−２０添加ＴＢＳ（TBS-Tween）溶液で２０００倍希釈した一次抗体とＰＶＤＦ膜を室温で２時間反応させた後、洗浄液で同様に３回洗浄した。二次抗体としてペルオキシダーゼで標識された抗マウスＩｇＧ又は抗ウサギＩｇＧ溶液 (Amersham Pharmacia Biotech UK, Buckinghamshire, UK)を洗浄液で５，０００倍希釈し、室温にて４０分間反応させ、同様に３回洗浄後、ＥＣＬ Western blotting detection reagents (Amersham Pharmacia Biotech UK社製)にて目的のタンパクを検出した。なお、抗ｐ５３(ａｂ-６)モノクローナル抗体はCALBIOCHEM社(Cambridge,MA)より、抗ｐｈｏｓｐｈｏ−Ｓｅｒ１５・ｐ５３ポリクローナル抗体はCell Signaling社(Beverly,MA)より購入した。ｐｉｆｉｔｈｒｉｎ−αはALEXIS社(Lausen, Switzerland)より購入した。結果を図３に示す。 After adding sample solution (50 mM Tris-HCl (pH 6.5), 10% glycerol, 2% SDS, 0.1% bromophenol blue, 40 mM dithiothreitol (DTT)) to protein extract, heat at 94 ° C for 3 minutes Denaturation was performed, and electrophoresis was performed using 12.5% e-PAGEEL (manufactured by ATTO Corporation) at a constant current of 36 mA in an electrophoresis buffer solution (20 mM Tris, 200 mM glycine, 0.3% SDS). The transferred protein was transferred to a polyvinylidene fluoride (PVDF) membrane (manufactured by Nippon Millipore Limited), and then room temperature in a 5% Tween-20 added TBS (TBS-Tween) solution in which 2.5% skim milk was dissolved. The PVDF membrane was shaken for 30 minutes. Thereafter, the PVDF membrane was washed with a TBS-Tween solution (cleaning solution) containing 0.25% skim milk for 10 minutes, 5 minutes, and 5 minutes. The primary antibody and PVDF membrane diluted 2000 times with 5% Tween-20-added TBS (TBS-Tween) solution were reacted at room temperature for 2 hours, and then washed three times in the same manner with a washing solution. Anti-mouse IgG or anti-rabbit IgG solution labeled with peroxidase as a secondary antibody (Amersham Pharmacia Biotech UK, Buckinghamshire, UK) was diluted 5,000 times with a washing solution, reacted at room temperature for 40 minutes, and washed three times in the same manner. Thereafter, the target protein was detected with ECL Western blotting detection reagents (Amersham Pharmacia Biotech UK). The anti-p53 (ab-6) monoclonal antibody was purchased from CALBIOCHEM (Cambridge, MA), and the anti-phospho-Ser15 / p53 polyclonal antibody was purchased from Cell Signaling (Beverly, MA). Pifithrin-α was purchased from ALEXIS (Lausen, Switzerland). The results are shown in FIG.
［結果］
図３に示されるように、ＡＬＡ存在下インビトロでＰＤＴを行った細胞において抗ｐ５３抗体と、抗ｐｈｏｓｐｈｏ−Ｓｅｒ１５ ｐ５３抗体が観察され、ｐ５３タンパクの安定化とｐｈｏｓｐｈｏ−Ｓｅｒ１５ｐ５３の発現は、ＡＬＡ存在下ＰＤＴを行った細胞においてのみ見られ、ｐｉｆｉｔｈｒｉｎ-αの存在下では、それらの発現は共に減少することが確認された。
[result]
As shown in FIG. 3, anti-p53 antibody and anti-phospho-Ser15 p53 antibody were observed in cells subjected to PDT in vitro in the presence of ALA. The stabilization of p53 protein and the expression of phospho-Ser15p53 were observed in the presence of ALA. It was observed only in the cells subjected to PDT, and it was confirmed that their expression decreases together in the presence of pifithrin-α.
（ＡＬＡ−ＰＤＴの殺細胞効果に対するｐ５３特異的阻害剤の影響）
３５ｍｍ培養シャーレ上にＭＫＮ−４５（２×１０５ｃｅｌｌｓ）を播種し、２４時間３７℃にて５％ＣＯ２インキュベーター内で培養した。ＡＬＡ塩酸塩を最終濃度０μＭ、５０μＭ、１００μＭ、２００μＭになるように添加し、さらに４時間から６時間培養を行った。ＤＬＤ−Ｒ２レーザー照射装置を用いて、２００ｍＷ、３分、３．７Ｊ／ｃｍ２相当を照射した。また、ｐｉｆｉｔｈｒｉｎ−αは最終濃度５０μＭを照射後に添加するもの、ＡＬＡ塩酸塩と同時に添加するもの、添加しないものの３群に分けた。照射７２時間後、培地を除去し、ｃｒｙｓｔａｌ ｖｉｏｌｅｔ溶液（0.5% crystal violet in 25% methanol）を５００μＬ添加し、室温で５分反応させた。Ｃｒｙｓｔａｌ ｖｉｏｌｅｔ溶液を捨て、流水で洗い、水を切った後５％デオキシコール酸ナトリウム溶液を１ｍＬ添加し、室温で１０分間反応させ、イムノリーダーを用いて５４０ｎｍで吸光度を測定し、実施例１と同様に、細胞増殖抑制率(％)を算出した。その結果、以下の表４に示すようにＡＬＡ塩酸塩と同時にｐｉｆｉｔｈｒｉｎ−αを添加した場合、他の２群と比較すると細胞増殖抑制率の低下が認められた。
(Effect of p53-specific inhibitor on cell killing effect of ALA-PDT)
MKN-45 (2 × 10 5 cells) was seeded on a 35 mm culture dish, and cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours. ALA hydrochloride was added to final concentrations of 0 μM, 50 μM, 100 μM, and 200 μM, and further cultured for 4 to 6 hours. Using a DLD-R2 laser irradiation apparatus, 200 mW, 3 minutes, 3.7 J / cm 2 equivalent was irradiated. Pifithrin-α was divided into three groups: those added at a final concentration of 50 μM after irradiation, those added simultaneously with ALA hydrochloride, and those not added. 72 hours after irradiation, the medium was removed, 500 μL of crystal violet solution (0.5% crystal violet in 25% methanol) was added, and the reaction was allowed to proceed at room temperature for 5 minutes. Discard the Crystal violet solution, wash with running water, drain the water, add 1 mL of 5% sodium deoxycholate solution, react for 10 minutes at room temperature, and measure the absorbance at 540 nm using an immunoreader. Similarly, the cell growth inhibition rate (%) was calculated. As a result, as shown in Table 4 below, when pifithrin-α was added at the same time as ALA hydrochloride, a decrease in the cell growth inhibition rate was observed compared to the other two groups.
Claims (15)
がん患者から採取した生体試料における、ＰＥＰＴ１（ペプチドトランスポーター１）の発現量の多寡を指標として、ＰＥＰＴ１の発現量が多いときに、５−アミノレブリン酸（ＡＬＡ）類がＡＬＡ−ＰＤＴ、及び／又は、ＡＬＡ−ＰＤＤに有効であると判定することを特徴とする有効性予測方法。 Predict the effectiveness of administering 5-aminolevulinic acid (ALA) prior to 5-aminolevulinic acid-photodynamic therapy (ALA-PDT) or 5-aminolevulinic acid-photodynamic diagnosis (ALA-PDD) A way to
When the expression level of PEPT1 is high, using a large amount of expression level of PEPT1 (peptide transporter 1) in a biological sample collected from a cancer patient as an indicator, 5-aminolevulinic acid (ALA) is ALA-PDT and / or Or the effectiveness prediction method characterized by determining with it being effective for ALA-PDD.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2009111948A JP2010259353A (en) | 2009-05-01 | 2009-05-01 | Method for predicting the effectiveness of 5-aminolevulinic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2009111948A JP2010259353A (en) | 2009-05-01 | 2009-05-01 | Method for predicting the effectiveness of 5-aminolevulinic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2010259353A true JP2010259353A (en) | 2010-11-18 |
Family
ID=43358001
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2009111948A Pending JP2010259353A (en) | 2009-05-01 | 2009-05-01 | Method for predicting the effectiveness of 5-aminolevulinic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2010259353A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04500770A (en) * | 1989-07-28 | 1992-02-13 | クイーンズ ユニバーシティ アット キングストン | Methods for detecting and treating malignant and non-malignant lesions by photochemotherapy |
-
2009
- 2009-05-01 JP JP2009111948A patent/JP2010259353A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04500770A (en) * | 1989-07-28 | 1992-02-13 | クイーンズ ユニバーシティ アット キングストン | Methods for detecting and treating malignant and non-malignant lesions by photochemotherapy |
Non-Patent Citations (5)
| Title |
|---|
| CSNC200900548014; 医学のあゆみ Ｖｏｌ．１８２， Ｎｏ．１, 19970705, ｐ．５８-６２ * |
| JPN6013060628; Ｌｉｖｅｒ Ｉｎｔ． Ｖｏｌ．２７， Ｎｏ．２, 20070219, ｐ．２０１-２０８ * |
| JPN6013060630; 医学のあゆみ Ｖｏｌ．１８２， Ｎｏ．１, 19970705, ｐ．５８-６２ * |
| JPN6013060633; Ｃａｎｃｅｒ Ｌｅｔｔ． Ｖｏｌ．２３９， Ｎｏ．２, 20051005, ｐ．１６８-１８２ * |
| JPN6013060636; Ｊ Ｐｈａｒｍａｃｏｌ Ｅｘｐ Ｔｈｅｒ． Ｖｏｌ．３１４， Ｎｏ．３, 20050518, ｐ．１０９３-１１００ * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dobashi et al. | Stepwise participation of p53 gene mutation during dedifferentiation of human thyroid carcinomas | |
| WO2014017491A1 (en) | Fusion gene of cep55 gene and ret gene | |
| CA2598510C (en) | Q3 sparc deletion mutant and uses thereof | |
| US20090136508A1 (en) | Method of diagnosing colon and gastric cancers | |
| EP2430452B1 (en) | Carcinoma diagnosis and treatments, based on odc1 genotype | |
| JP2008118915A (en) | Diagnosis of gastric cancer by identification of high expression gene of gastric cancer and its use for drug discovery | |
| JP2010248210A (en) | Disease imaging, diagnosis, and treatment | |
| JP2004518429A (en) | Method for determining prescription by chemotherapy based on ERCC1 expression | |
| Bhuvaneswari et al. | Targeting EGFR with photodynamic therapy in combination with Erbitux enhances in vivo bladder tumor response | |
| Rajagopalan et al. | The Stress-response protein prostate-associated gene 4, interacts with c-Jun and potentiates its transactivation | |
| Ghahe et al. | Increased DNA repair capacity augments resistance of glioblastoma cells to photodynamic therapy | |
| US20170051358A1 (en) | Compositions and methods for identification, assessment, prevention, and treatment of cancer using nfs1 biomarkers and modulators | |
| TW200418988A (en) | Method for diagnosing prostate cancer | |
| JP2004507254A (en) | Diagnosis and treatment of cancer | |
| JP2005514918A (en) | Phosphatase associated with metastasis | |
| JP7025034B2 (en) | Compositions and Methods for Treating and Preventing Pancreatitis, Kidney Injury and Kidney Cancer | |
| KR20060048684A (en) | How to diagnose and treat cancer | |
| KR101860652B1 (en) | Markers for diagnosing of gastrointestinal stromal tumors and method for diagnosing of gastrointestinal stromal tumors | |
| JP2010259353A (en) | Method for predicting the effectiveness of 5-aminolevulinic acid | |
| CN111139299B (en) | Application of JOSD2 protein in preparing medicine for treating malignant tumor | |
| JP2007528717A (en) | How to diagnose colorectal cancer | |
| FR2954352A1 (en) | PREDISPOSITION MARKER FOR CANCER | |
| KR20050004076A (en) | Methods for assessing and treating cancer | |
| JP4904269B2 (en) | Methods for screening carnitine transporter agonists or antagonists and uses thereof | |
| Kong et al. | Alterations of the tumour suppressor gene DCC in neuroblastoma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20120412 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20131209 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140130 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20140901 |