JP5435461B2 - Migration inhibitor for cancer treatment - Google Patents

Description

  The present invention relates to a migration inhibitor that suppresses or inhibits the enhancement of tumor cell migration in cancer treatment using radiation or the like.

  Radiation is an important therapeutic tool in the treatment of solid cancer, but clinically, recurrence of tumors within and outside the field is often observed. It has been explained that the cause is that the micro-metastasis already exists before irradiation, or that the irradiation field and irradiation dose do not reach such a level that a sufficient effect can be obtained.

  In clinical settings, high-dose irradiation over a wide area increases the possibility of causing brain necrosis, while if the dose is kept low, local recurrence in the irradiation field is essential. Recently, 90 Gy equivalent irradiation was applied to the tumor mass, and a new treatment device such as Tomotherapy (registered trademark), which is an intensity-modulated irradiation method that can weight 60 Gy to the surrounding infiltration site and 40-50 Gy to the surrounding brain, is used. In addition, the conventional external irradiation is combined with a gamma knife and a cyber knife.

  Current radiation therapy can be said to be the result of exploration obtained from clinical experience of radiation fields and radiation methods that determine the safe amount that does not cause brain necrosis and maximize the therapeutic effect. As a new radiation source, the application of heavy particle beams has been started.

  However, treatments for undifferentiated tumors, malignant melanoma, malignant brain tumors, and glioblastomas that occur in major organs such as the lungs, digestive organs, gynecological organs, breasts, liver, etc. have been sufficiently effective. The situation that cannot be said continues. In these solid cancers exhibiting invasive growth, not only local recurrence after surgery, but also remote metastasis to brain organs and brain metastasis are not rare and have become a serious clinical problem.

  Various attempts have been made for glioblastoma, which has a particularly high grade of malignancy, but it has not reached a long-term survival period. Attempts at 90 Gy high-dose irradiation have been reported for glioblastoma, but the mean median survival was 16.2 months, and the frequency of cerebrospinal fluid seeding increased compared to the standard-dose irradiation group (Non-Patent Documents) 1). The current status of treatment of 22 cases of glioblastoma with carbon beam alone, a kind of heavy particle beam, has a median average survival of 10-11 months, and it is still possible to achieve safe radiation dose and maximum therapeutic effect. Searching for irradiation field, irradiation method (number of divisions, etc.), temozolomide combination, etc. (29th Central Neurological Tumor Clinical Research Group Meeting Central Nervous System II (0101) Group Meeting Report Date: September 8, 2008 Day).

  For these reasons, refractory cancer cannot be cured simply by increasing the irradiation dose, and even when a new treatment device, a heavy particle beam, can be locally controlled by single treatment, control of extra-irradiation metastasis and distant metastasis It is difficult to achieve a significant prognosis, and as a result, it is becoming impossible to conquer cancers that show invasive growth.

  Overcoming refractory cancer has been a problem to be solved for a long time as the aging of society progresses and the cancer population increases, and it is a serious problem with urgency. The refractory treatment of refractory cancer by radiation has been described as radiation resistance of tumor cells, but recently, the biological effects of radiation have been analyzed, and radiation itself is dedifferentiation of tumor cells. There have been reports on the promotion of migration or increased migration (Non-patent Documents 2-4), and the hypothesis of molecular mechanisms explaining the background of irradiation resistance. Specifically, hypothesis that DNA repair enzyme is activated by irradiation (Non-patent document 5), and specific activation of epithelial growth factor of irradiated cells and (Non-patent document 6) phosphorylated inositol triphosphate pathway (Non-Patent Document 7), but it has not been linked to specific clinical results. Searching for prior art and analogy only from past literature, it is difficult to elucidate the molecular mechanism that explains the enhancement of migration and invasion due to irradiation, and it still remains unsolved.

  On the other hand, the present inventor expressed a calcium permeable AMPA receptor among glutamate receptors in glioblastoma cells, and a slow increase in intracellular calcium concentration through this channel caused proliferation and migration of tumor cells. It has been found to promote (Non-Patent Documents 8 and 9).

  Then, the present inventor has proceeded with studies on AMPA receptors and proved in an in vivo experimental system using nude mice that the antagonist can suppress tumor cell growth and be a therapeutic agent for glioblastoma. Have proposed an AMPA receptor antagonist as a “glioblastoma therapeutic agent” (Patent Document 1).

  In addition, the present inventors have found that GluR2 DNA that converts calcium-permeable AMPA receptor channel expressed in glioblastoma cells to impermeability suppresses infiltration and proliferation of glioblastoma cells, and It has been proposed that the incorporated adenovirus vector is useful as a gene therapy agent for glioblastoma (Patent Document 2).

  Furthermore, the present inventor observed the irradiated and non-irradiated cells with a time-lapse microscope for a long period of time using a human glioblastoma cell model that has been uniquely established, thereby allowing cytoplasmic division and migration (migration) in glioma cells. ) Is a related series of phenomena. More specifically, a compound having a cytokinesis-promoting substance production-suppressing activity suppresses or inhibits the enhancement of migration of tumor cells associated with radiation in cancer treatment by irradiation with radiation such as heavy particle beams. And has been found to provide a means for inhibiting radiosensitization of migration (Patent Document 3).

International Publication WO2003 / 082332 Pamphlet JP 2004-67627 A Japanese Patent Application No. 2007-127361

Lancet Oncology 6, 953-960 (2005). J. Radiat. Res. (Tokyo) 46, 43-50 (2005). Eur. J. Cancer 43, 1214-1224 (2007). Cancer Res. 61, 2744-2750 (2001). Nature 444, 756-760 (2006). Mol. Cancer Res. 6,996-1002 (2008). J. Neurooncol 76, 227-237 (2006). Nature Med. 8,971-978 (2002). J. Neurosci. 27, 7987-8001 (2007).

  The present inventor has proceeded with studies on AMPA type receptors that are involved in fast neurotransmission, a kind of such ionic glutamate receptors, and also promoted the phenomenon of increased cell migration (migration) in irradiated glioma cells. During the analysis process, when the tumor mass is irradiated, tumor cells that are not directly irradiated from the tumor mass one after another migrate at a high speed, and a part of the tumor mass is irradiated using a microbeam irradiation device. In addition, since the migration of the entire tumor mass is induced, it was found that the cell migration phenomenon was also enhanced in non-irradiated cells around the irradiated cells.

  This phenomenon does not occur when the culture dish is emptied using a microbeam irradiation device. Therefore, it is thought that the irradiated cells give some signal and this signal induces the promotion of tumor cell migration. It was. Using a diaminofluorescein-2 diacetate (DAF-2 DA), a nitric oxide (NO) fluorescent probe, capture the fluorescence of DAF-2T reacted with NO in the cell using a confocal microscope In addition to NO acting as an intercellular signal for increased migration, NO promoted migration of AMPA-type receptor GluR1 subunit to the cell membrane, leading to an increase in intracellular calcium and cell migration. Found to promote.

That is, it was found that the NO signal emitted from the irradiated cells is important for the local movement of GluR1 (see the conceptual diagram in FIG. 1). When live staining is performed with an antibody that recognizes the extracellular domain of GluR1, GluR1 is actually trafficked to the cell membrane by NO. In addition, cell surface proteins were cross-linked using BS ³ (bis [sulfosuccinimidyl] suberate, Pierce Biotechnology, Rockford, IL 61105 USA), a crosslinker solution, and then quenched with 1M glycine to receive the cell surface. When the body and cytoplasmic receptors were separated and analyzed, it was found that irradiation promotes trafficking of the GluR1 receptor to the cell membrane.

  Based on this finding, it was confirmed that when the intracellular calcium concentration of irradiated cells reacting with AMPA and cyclothiazide (CTZ) was measured by calcium photometry, it was enhanced twice that of non-irradiated cells. When irradiating a single cell strictly with a microbeam device, it was confirmed that NO was propagated between cells, and then GluR1 was further interfered with RNA to reduce the expression. When this was done, the intercellular propagation of NO disappeared, suggesting that there was a bidirectional signal of NO-GluR1 in the background of enhanced migration of irradiated cells and increased migration of surrounding non-irradiated cells.

  Combining NO neutralizing agents, NOS inhibitors, and AMPA receptor antagonists with irradiation suppresses NO production, controls trafficking of GluR1 to the membrane, and stops migration promotion by irradiation. The specific migration inhibitory effect of this combination with irradiation of a compound having a nitric oxide production-suppressing activity and a neutralizing activity is completely unexpected and unpredictable from conventional knowledge and the inventor's own previous findings. That is.

  The present invention has been made in view of the circumstances as described above, and has to be solved for solid cancers. For metastasis, infiltration, and seeding, a radiosensitizer, a cancer therapeutic agent that also functions as a migration inhibitor, and cancer metastasis. The problem is to provide a preventive agent.

  The present invention is characterized by the following in order to solve the above problems.

  1: A radiosensitizing migration inhibitor for radiation cancer treatment, comprising as an active ingredient a compound having activity of inhibiting production of nitric oxide (NO) and nitric oxide synthase (NOS).

  Second: The first radiosensitizing migration inhibitor for radiation cancer treatment according to the first aspect, wherein the compound has at least iNOS production inhibitory activity.

Third: the compound is ebselen edaravone
Pravastatin Na (trade name Mevalotin)
Simvastatin (trade name Lipobas)
Fluvastatin Na (trade name Law Coal)
Atorvastatin Ca hydrate (trade name Lipitor)
Pitavastatin Ca (trade name Rivaro)
The radiosensitizing migration inhibitor for the first or second radiation cancer treatment, which is any compound selected from the group consisting of:

Fourth: the compound is 1400W
[N- (3-Aminomethyl) benzylacetamidine, 2HCl]
1-Amino-2-hydroxyguanidine, p -Toluenesulfonate
Aminoguanidine, Hemisulfate
Angeli's Salt (AS; Disodium Diazen-1-ium-1,2,2-triolate; Sodium α-Oxyhyponitrite; Sodium Trioxodinitrate)
Bromocriptine Mesylate (BCT; BRC; 2-Bromo-α-ergocryptine, Methanesulfonate)
NG, NG´-Dimethyl-L-arginine, Dihydrochloride (SDMA, 2HCl)
NG, NG-Dimethyl-L-arginine, Dihydrochloride (ADMA, 2HCl)
Diphenyleneiodonium Chloride (DPI)
2-Ethyl-2-thiopseudourea, Hydrobromide (S-Ethyl-ITU, HBr; S-Ethylisothiourea, HBr)
L-Thiocitrulline, Dihydrochloride (2-Thioureido-L-norvaline)
MEG, Hydrochloride (Mercaptoethylguanidine, HCl)
NG-Monomethyl-D-arginine, Monoacetate Salt (NG-Me-D-Arg, AcOH; Nω-Me-D-Arg; D-NMMA)
NG-Monomethyl-L-arginine (L-NMMA)
NG-Monomethyl-L-arginine, Monoacetate (Nω-Me-L-Arg; NG-Me-L-Arg, AcOH; L-NMMA)
L-NIL, Dihydrochloride [LN ^6- (1-Iminoethyl) lysine, DiHCl]
7-Nitroindazole, Sodium Salt (7-NiNa)
7-Nitroindazole, 3-Bromo-, Sodium Salt (BrNINa)
{(4S) -N- (4-Amino-5 [aminoethyl] aminopentyl) -N´-nitroguanidine, TFA}
LN ^5- (1-Iminoethyl) ornithine, HCl
S-Methyl-L thiocitrulline, HCl
NG-Monomethyl-L-arginine Monoacetate Salt
NG-Nitro-L-arginine Methyl Ester, HCl
NG-Nitro-D-arginine Methyl Ester, HCl
7-Nitroindazole
L-Thiocitrulline, HCl
Nα-Tosyl-Phe Chloromethyl Ketone (TPCK)
1,3-PBITU, Dihydrobromide [S, S´-1,3-Phenylene-bis (1,2-ethanediyl) -bis -isothiourea, 2HBr]
NG-Propyl-L-arginine (N-PLA; Nω-Propyl-L-arginine)
PTIO (2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide)
SIN-1, Hydrochloride (3-Morpholinosydnonimine, HCl)
S-Methylisothiourea, Sulfate (2-Methyl-2-thiopseudourea, Sulfate; SMT)
L-NMMA
S-Methyl-L-thiocitrulline, Dihydrochloride [Nδ- (S-Methyl) isothioureido-L-ornithine]
Sodium Nitroprusside, Dihydrate [SNP; Sodium Nitroferricyanide (III) Dihydrate]
TRIM [1- (2-Trifluoromethylphenyl) imidazole]
Zinc (II) Protoporphyrin IX (ZnPP-9)
NG-Nitro-L-arginine (L-NNA; NG-NO ₂ -L-Arg)
NG-Nitro-L-arginine Methyl Ester, Hydrochloride (L-NAME, HCl; Nω-NO ₂ -L-Arg-OMe; NG-NO ₂ -L-Arg-OMe)
LY 83583 (6-Anilino-5,8-quinolinequinone)
ODQ (1H- [1,2,4] Oxadiazolo [4,3-a] quinoxalin-1-one)
S-Methyl-ITU
S-Ethyl-ITU
S-Isopropyl-ITU
S-Aminoethyl-ITU
2-Iminopiperidine
DAHP
7-Nitroindazole
3-Bromo-7- Nitroindazole
APDC
The radiosensitizing migration inhibitor for the first or second radiation cancer treatment, which is any compound selected from the group consisting of:

  Fifth: A migration inhibitor for cancer treatment comprising a compound having activity of inhibiting production of nitric oxide (NO) and nitric oxide synthase (NOS) as an active ingredient.

  Sixth: The fifth migration inhibitor for cancer treatment, wherein the compound has at least iNOS production inhibitory activity.

Seventh: The compound is ebselen edaravone (trade name Rajcut)
Pravastatin Na (trade name Mevalotin)
Simvastatin (trade name Lipobas)
Fluvastatin Na (trade name Law Coal)
Atorvastatin Ca hydrate (trade name Lipitor)
Pitavastatin Ca (trade name Rivaro)
The migration inhibitor for treating the fifth or sixth cancer, wherein the compound is any compound selected from the group consisting of:

Eighth: The compound is 1400W
[N- (3-Aminomethyl) benzylacetamidine, 2HCl]
1-Amino-2-hydroxyguanidine, p -Toluenesulfonate
Aminoguanidine, Hemisulfate
Angeli's Salt (AS; Disodium Diazen-1-ium-1,2,2-triolate; Sodium α-Oxyhyponitrite; Sodium Trioxodinitrate)
Bromocriptine Mesylate (BCT; BRC; 2-Bromo-α-ergocryptine, Methanesulfonate)
NG, NG´-Dimethyl-L-arginine, Dihydrochloride (SDMA, 2HCl)
NG, NG-Dimethyl-L-arginine, Dihydrochloride (ADMA, 2HCl)
Diphenyleneiodonium Chloride (DPI)
2-Ethyl-2-thiopseudourea, Hydrobromide (S-Ethyl-ITU, HBr; S-Ethylisothiourea, HBr)
L-Thiocitrulline, Dihydrochloride (2-Thioureido-L-norvaline)
MEG, Hydrochloride (Mercaptoethylguanidine, HCl)
NG-Monomethyl-D-arginine, Monoacetate Salt (NG-Me-D-Arg, AcOH; Nω-Me-D-Arg; D-NMMA)
NG-Monomethyl-L-arginine (L-NMMA)
NG-Monomethyl-L-arginine, Monoacetate (Nω-Me-L-Arg; NG-Me-L-Arg, AcOH; L-NMMA)
L-NIL, Dihydrochloride [LN ^6- (1-Iminoethyl) lysine, DiHCl]
7-Nitroindazole, Sodium Salt (7-NiNa)
7-Nitroindazole, 3-Bromo-, Sodium Salt (BrNINa)
{(4S) -N- (4-Amino-5 [aminoethyl] aminopentyl) -N´-nitroguanidine, TFA}
LN ^5- (1-Iminoethyl) ornithine, HCl
S-Methyl-L thiocitrulline, HCl
NG-Monomethyl-L-arginine Monoacetate Salt
NG-Nitro-L-arginine Methyl Ester, HCl
NG-Nitro-D-arginine Methyl Ester, HCl
7-Nitroindazole
L-Thiocitrulline, HCl
Nα-Tosyl-Phe Chloromethyl Ketone (TPCK)
1,3-PBITU, Dihydrobromide [S, S´-1,3-Phenylene-bis (1,2-ethanediyl) -bis -isothiourea, 2HBr]
NG-Propyl-L-arginine (N-PLA; Nω-Propyl-L-arginine)
PTIO (2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide)
SIN-1, Hydrochloride (3-Morpholinosydnonimine, HCl)
S-Methylisothiourea, Sulfate (2-Methyl-2-thiopseudourea, Sulfate; SMT)
L-NMMA
S-Methyl-L-thiocitrulline, Dihydrochloride [Nδ- (S-Methyl) isothioureido-L-ornithine]
Sodium Nitroprusside, Dihydrate [SNP; Sodium Nitroferricyanide (III) Dihydrate]
TRIM [1- (2-Trifluoromethylphenyl) imidazole]
Zinc (II) Protoporphyrin IX (ZnPP-9)
NG-Nitro-L-arginine (L-NNA; NG-NO ₂ -L-Arg)
NG-Nitro-L-arginine Methyl Ester, Hydrochloride (L-NAME, HCl; Nω-NO ₂ -L-Arg-OMe; NG-NO ₂ -L-Arg-OMe)
LY 83583 (6-Anilino-5,8-quinolinequinone)
ODQ (1H- [1,2,4] Oxadiazolo [4,3-a] quinoxalin-1-one)
S-Methyl-ITU
S-Ethyl-ITU
S-Isopropyl-ITU
S-Aminoethyl-ITU
2-Iminopiperidine
DAHP
7-Nitroindazole
3-Bromo-7- Nitroindazole
APDC
The migration inhibitor for treating the fifth or sixth cancer, wherein the compound is any compound selected from the group consisting of:

  According to the present invention, there is provided a radiosensitizing migration inhibitor capable of suppressing or inhibiting the enhancement of migration of tumor cells associated with radiation in cancer treatment by irradiation with radiation including heavy particle beams. Furthermore, it is also possible to suppress or inhibit the enhancement of tumor cell migration even in general cancer treatment with drugs or the like.

It is the figure which showed the conceptual diagram of the molecular mechanism of acceleration | stimulation of the radiation-induced invasive proliferation via nitric oxide obtained from the experiment using a clinical specimen. Increased migration of tumor cells by irradiation is triggered by the expression of NO due to the involvement of Akt and iNOS (NOS2), and the accumulated NO promotes the movement of GluR1 subunits to the cell membrane, resulting in an increase in intracellular calcium. Due to enhancement. It is the figure which showed the result of the migration ability evaluation using a clinical sample. From the experimental results on cell migration of X-ray and particle irradiation using dispersion culture (aggregation culture), non-irradiated cells also run radially at higher speed than aggregates. It was suggested that the signal of cell migration enhancement might be issued. Focusing on Nitric Oxide (NO), which is a non-structural gas, high-dose irradiation 10Gy is applied to aggregates, and staining with NO detection agents Diaminofluorescein-2 Diacetate (DAF-2 DA) and Mitotracker is performed for 4 hours after irradiation. Trials were performed at 8, 15, 24, 30 and 35 hours (n = 3). In addition, the migration velocity was analyzed for 35 hours immediately after irradiation with a time-lapse microscope. The experimental results showed similar results for X-ray and particle-irradiated cells. In other words, migratory cells around the agglomerate became NO positive after 4 hours of irradiation, cells around the tumor mass became positive after 8-15 hours, and the tumor mass became strongly positive after 15-24 hours, high The NO positive image continued until 35 hours later. NO (2-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO), 20-100μM, the dyeability of DAF-2 DA addition At the same time, enhanced cell migration by irradiation was stopped, and the migration rate was reduced to the level of non-irradiated cells. The figure shows live staining with DAF-2T (green) and Mitotracker (red) 10 Gy and 15 h after carbon beam irradiation, followed by fixation with 4% paraformaldehyde and observation with a confocal microscope. Spindle type cells that migrate around the agglomerate are DAF-2T positive (green), and DAF-2T positive (green) migrating cells are markedly reduced in the NO neutralizer cPTIO administration group. DAF-2T (green), Mitotracker (red), and DAPI (blue) stained images after 35 hours of X-ray irradiation show NO in contrast to the spindle-shaped cells migrating from the aggregates in a spiny manner No migrating cells appearing from the aggregates were observed in the Japanese cPTIO administration group. Bar; 100 μm. It is the figure which showed the result of the migration suppression (dispersion culture) by NO neutralizer (20 micromol cPTIO) using a clinical specimen. Implantation of 10 Gy X-rays on the graft culture and simultaneous addition of NO neutralizer (20 μM cPTIO in the medium decreased the migration rate to the non-irradiated group level. The figure shows every hour from 12 hours to immediately after irradiation. The time-lapse image of tumor cells treated with NO neutralizer was 8.5 ± 3.2 μm / h for 12h, 12.5 ± 4.2 μm / h for the next 24h (n = 3), treated with NO neutralizer 21.5 ± 5.7 μm / h during the first 12 h (n = 6), 23.5 ± 4.7 μm / h for the next 24h (n = 3). * P <0.05, Bar; 100 μm. It is the figure which showed the result of the migration ability evaluation using a clinical sample. It shows that NO production by irradiation is induced by NOS2. Irradiate 10Gy X-rays, seed cells in cloning ring and irradiate 6 hours after culturing, transfection with siRNA of cPTIO, 20μM and 100μM, L-NAME, 1mM, NOS1, NOS2, and NOS3, and RNA interference. Remove ring after time culture (white is the border of ring), then administer DAF-2DA after further culture for 24 hours (NO expressing cells turn to DAF-2T and turn green), fix, and nuclear staining with DAPI (blue) did. cPTIO and NOS2 RNA interference and migration inhibition were observed in the L-NANE group. * P <0.01, ** p <0.001, Bar; 100 μm. It is the figure which showed the result of the migration ability evaluation using a clinical sample. It shows that the production of NO by irradiation is suppressed by RNA interference with Akt1. Irradiated with 10 Gy X-rays, seeded cells in cloning ring, non-irradiated control after 6 hours of culture, irradiated with 10 Gy X-ray, administered cPTIO, 20 μM, GYKI52466 only for 1 h just before 100 μM irradiation and transfection with Akt1 siRNA to interfere with RNA, and further 12 Remove ring after time culture (white is the border of ring), then administer DAF-2DA after further culture for 24 hours (NO expressing cells turn to DAF-2T and turn green), fix, and nuclear staining with DAPI (blue) did. In the 10GyX-irradiated group, the increase of NO production and the increase of migratory cells and phosphorylation of Akt Ser 473 are promoted. In the cPTIO, GYKI52466, and Akt1 RNA interference groups, decreased NO production, inhibition of migration, and dephosphorylation of Akt Ser473 were observed. Bar; 100 μm. It is the figure which showed evaluation of intracellular calcium concentration by irradiation using a clinical specimen. Fluo 3-AM is loaded onto cells, excited at 488 nm, and 200 μM AMPA and 100 μM CTZ are administered to irradiated and non-irradiated cells to measure intracellular calcium concentration ([Ca ²⁺ ] _i ) induced by AMPA did. Irradiation changed [Ca ²⁺ ] _i and increased it about twice. It is the figure which showed that the movement to the cell membrane of GluR1 by irradiation using a clinical sample is induced in cGMP dependence. Indirect fluorescent staining was performed using an antibody that recognizes the N-terminal of GluR1. Cells were fixed for 1 minute with 4% PFA on the surface only. Various drugs of cells were administered. The administration concentration and exposure time are shown in parentheses. NOC-18 (1mM, 24h), NOC18 (1mM, 24h) + cPTIO (100 μM, 24h), NOC18 (1mM, 24h) + cPTIO (100μM, 24h) + 8Br-cGMP (500μM, 24h), 8Br-cGMP (500μM, 2h), 8Br-cGMP (500μM, 2h) + cPTIO (100μM, 2h), 8Br-cGMP (500μM, 2h) + KT5823 (10μM, 2h), RT10Gy, RT10Gy + KT5823 (10μM, 24h), RT10Gy + cPTIO (100 μM, 24 h), and RT10Gy + cPTIO (100 μM, 24 h) + 8Br-cGMP (500 μM, 24 h). The suface expression of GluR1 was quantified (mean ± SEM; n = 4.). It is the figure which showed that the localization of the movement GluR1 which carried out the cell membrane by irradiation using a clinical specimen, and cGKII are co-expressing. GluR1 surface expression and cGKII stained image. Upper row: X-ray 10Gy irradiated cells. Bottom: non-irradiated cells. It is the figure which showed cross-linking assay that the localization of cell membrane GluR1 increases by irradiation using a clinical specimen. GluR1 crosslinking assay using 10Gy X-ray irradiated and non-irradiated cells. Radiation doubles the membrane surface receptors. In non-irradiated cells, the cytoplasmic component and the membrane component were halved, but in irradiated cells, 3/4 of the entire receptor was found to be a membrane component. * p <0.01 FIG. 4 shows that Ser845 phosphorylation of GluR1 is induced by irradiation using a clinical sample, and this phosphorylation is suppressed by the NO neutralizer cPTIO. Using an antibody that recognizes phosphorylation at the Ser845 site of GluR1, 10 Gy X-ray irradiation, 10 Gy X-ray irradiation treated with 100 μM cPTIO and non-irradiated cells were immunoblotted. For quantification, the phosphorylation signal of each group was normalized with total GluR1, and the obtained value was further normalized with respect to the control (* p <0.05, n = 4). It is the figure which showed that the propagation of NO was suppressed by inhibiting a calcium-permeable AMPA type | mold receptor using a clinical sample, and the migration enhancement induced by irradiation stopped. In the figure, “a” shows that migration inhibition occurs due to interference with an AMPA receptor antagonist and GRIA1 by siRNA. Ability to test for migration by Ring -proliferation test. 10Gy X-ray cells were treated with flurescently-labelled RNA (red) and used as transfection indicators (left figure). In order to knock down GluR1, GRIA1 was interfered with siRNA and simultaneously treated with flurescently-labelled RNA (red), which was used as an index for transfection (center). 100 μM GYKI52466 was administered 1 hour before irradiation, removed immediately after irradiation, and replaced with medium. The distribution range of NO was confirmed with a confocal microscope that DAF-2DA was added to cells and DAF-2 was converted to DAF-2T and emitted green fluorescence in the presence of NO. The white line is the ring boundary. In the figure, b indicates that NO propagates to cells that are not irradiated even when irradiated with a single cell. Using a microbeam irradiation device, a single cell (differential interference image, right figure) is irradiated with 5 neon ions (inset, right figure) on the cytoplasm, and NO propagation is achieved by DAF-2T fluorescent (green) (middle figure). What I saw. The white arrows in the middle and right figures indicate the same cells. Cells are stained with fluroescently-labelled RNA (red, left figure) and DNA stained with DAPI (not shown). Transfection efficiency is over 98%. In the figure, c shows that when GluR1 is knocked down, NO does not propagate even when a single cell is irradiated. Using a microbeam irradiation device, 1 cell (differential interference image, right figure) is irradiated with 5 neon ions (inset, right figure) on the cytoplasm, and NO propagation is observed with DAF-2T fluorescent (green) (center figure). Things. The white arrows in the middle and right figures indicate the same cells. The cells are fluroescently-labelled RNA and GRIA1 knocked down with siRNA (red, left figure) and DNA stained with DAPI (not shown). Transfection efficiency is over 98%. It is the figure which showed the protein expression of GluR1, iNOS, and cGKII in the invasive growth most advanced region using the surgically-extracted specimen of the patient who performed radiation therapy. In the figure, “a” shows a typical pathological finding of the infiltrating most distal portion after irradiation, and shows a typical finding of the excised tissue after irradiation. Massive necrosis (N) and hyalinized microvascular proliferations (V) are observed. Small round cells (red arrows) and spindles (blue arrows) infiltrate the surrounding brain tissue. H.E. Stained image. In the figure, b shows the immunohistological findings of the small round cells and spindle cells at the invasion frontmost part. Double-stained the infiltrating cutting edge (left figure, white frame) with vimentin (VIM) and GluR1 (upper right figure), and the black frame in the left figure with iNOS and cGKII (lower right figure) .

  Hereinafter, the present invention will be described in detail.

High line energy imparted (LET) charged particle beams such as ¹² C, ²⁰ Ne, and ⁴⁰ Ar ion beams have relatively high biological activity compared to low LET X-rays. Therefore, the most undifferentiated and invasive human cancers such as glioblastoma cells are usually resistant to conventional X-ray therapy, so a new technique called high-LET radiation therapy is used to treat these cancers. Promising. In addition, the high-LET charged particle beam has a sharp Bragg peak, so the spatial distribution is more accurate and the localization is well defined, so it is possible to treat lesions while minimizing adverse effects on surrounding anatomy. It becomes.

High line energy imparted (LET) charged particle beams such as ¹² C ion beam, ²⁰ Ne ion beam, and ⁴⁰ Ar ion beam have the potential to become new therapies for the treatment of X-ray resistant cancer, In the present invention, it has been confirmed that the radioactivity of the tumor is determined by the radiosensitivity to both tumor cell proliferation and metastasis. High LET radiation has a marked cytotoxic effect on CGNH-89, a human glioblastoma cell line. ^The relative biological effects of ¹² C, ²⁰ Ne, and ⁴⁰ Ar ion beams on X-rays were calculated as D ₁₀ , the dose representing 10% viability of cloneable cells, respectively, 3.4, 4.5 and 6.2. I became Gy. A single massive 10 Gy dose of high and low LET radiation promotes tumor cell metastasis.

As specific examples of compounds (hereinafter referred to as NO, NOS inhibitors) having activity of inhibiting production of nitric oxide (NO) and nitric oxide synthase (NOS) used as active ingredients in the present invention,
Ebselen edarabon (trade name Rajcut)
Pravastatin Na (trade name Mevalotin)
Simvastatin (trade name Lipobas)
Fluvastatin Na (trade name Law Coal)
Atorvastatin Ca hydrate (trade name Lipitor)
Pitavastatin Ca (trade name Rivaro)
Etc. These substances can be produced by referring to synthesis methods described in known literature or by using ordinary synthesis methods, and can also be obtained from the manufacture, sales, development companies, etc. of these substances. it can.

Moreover, the following can be mentioned as selective NO and NOS inhibitors.
1400W
[N- (3-Aminomethyl) benzylacetamidine, 2HCl]
1-Amino-2-hydroxyguanidine, p -Toluenesulfonate
Aminoguanidine, Hemisulfate
Angeli's Salt (AS; Disodium Diazen-1-ium-1,2,2-triolate; Sodium α-Oxyhyponitrite; Sodium Trioxodinitrate)
Bromocriptine Mesylate (BCT; BRC; 2-Bromo-α-ergocryptine, Methanesulfonate)
NG, NG´-Dimethyl-L-arginine, Dihydrochloride (SDMA, 2HCl)
NG, NG-Dimethyl-L-arginine, Dihydrochloride (ADMA, 2HCl)
Diphenyleneiodonium Chloride (DPI)
2-Ethyl-2-thiopseudourea, Hydrobromide (S-Ethyl-ITU, HBr; S-Ethylisothiourea, HBr)
L-Thiocitrulline, Dihydrochloride (2-Thioureido-L-norvaline)
MEG, Hydrochloride (Mercaptoethylguanidine, HCl)
NG-Monomethyl-D-arginine, Monoacetate Salt (NG-Me-D-Arg, AcOH; Nω-Me-D-Arg; D-NMMA)
NG-Monomethyl-L-arginine (L-NMMA)
NG-Monomethyl-L-arginine, Monoacetate (Nω-Me-L-Arg; NG-Me-L-Arg, AcOH; L-NMMA)
L-NIL, Dihydrochloride [LN ^6- (1-Iminoethyl) lysine, DiHCl]
7-Nitroindazole, Sodium Salt (7-NiNa)
7-Nitroindazole, 3-Bromo-, Sodium Salt (BrNINa)
{(4S) -N- (4-Amino-5 [aminoethyl] aminopentyl) -N´-nitroguanidine, TFA}
LN ^5- (1-Iminoethyl) ornithine, HCl
S-Methyl-L thiocitrulline, HCl
NG-Monomethyl-L-arginine Monoacetate Salt
NG-Nitro-L-arginine Methyl Ester, HCl
NG-Nitro-D-arginine Methyl Ester, HCl
7-Nitroindazole
L-Thiocitrulline, HCl
Nα-Tosyl-Phe Chloromethyl Ketone (TPCK)
1,3-PBITU, Dihydrobromide [S, S´-1,3-Phenylene-bis (1,2-ethanediyl) -bis -isothiourea, 2HBr]
NG-Propyl-L-arginine (N-PLA; Nω-Propyl-L-arginine)
PTIO (2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide)
SIN-1, Hydrochloride (3-Morpholinosydnonimine, HCl)
S-Methylisothiourea, Sulfate (2-Methyl-2-thiopseudourea, Sulfate; SMT)
L-NMMA
S-Methyl-L-thiocitrulline, Dihydrochloride [Nδ- (S-Methyl) isothioureido-L-ornithine]
Sodium Nitroprusside, Dihydrate [SNP; Sodium Nitroferricyanide (III) Dihydrate]
TRIM [1- (2-Trifluoromethylphenyl) imidazole]
Zinc (II) Protoporphyrin IX (ZnPP-9)
NG-Nitro-L-arginine (L-NNA; NG-NO ₂ -L-Arg)
NG-Nitro-L-arginine Methyl Ester, Hydrochloride (L-NAME, HCl; Nω-NO ₂ -L-Arg-OMe; NG-NO ₂ -L-Arg-OMe)
LY 83583 (6-Anilino-5,8-quinolinequinone)
ODQ (1H- [1,2,4] Oxadiazolo [4,3-a] quinoxalin-1-one)
S-Methyl-ITU
S-Ethyl-ITU
S-Isopropyl-ITU
S-Aminoethyl-ITU
2-Iminopiperidine
DAHP
7-Nitroindazole
3-Bromo-7- Nitroindazole
APDC
Cells exposed to radiation after 1 hour exposure to these substances have been shown to significantly promote inhibition of cell proliferation and cell motility. These results confirm that new radiation therapy should be combined with the administration of cell migration inhibitors.

  In the present invention, the radiosensitizing migration inhibitor for the treatment of radiation cancer as described above is provided, and the migration inhibitor for other cancer treatment by a drug or the like is also provided.

  The preparation of the migration inhibitor according to the present invention is prepared using carriers, excipients and other additives that are usually used for formulation. The carrier or excipient for the preparation may be either solid or liquid, such as lactose, magnesium stearate, starch, talc, gelatin, agar, pectin, gum arabic, olive oil, sesame oil, cocoa butter, Examples include ethylene glycol and other commonly used ones.

  Administration can be in any form of oral administration by tablets, pills, capsules, granules, powders, liquids, etc., or parenteral administration by injections such as intravenous injection, intramuscular injection, suppositories, and transdermal. Also good.

  In the treatment of radiation cancer, for example, the use of NO and NOS inhibitors is given by oral administration or injection immediately before daily irradiation in the case of fractional irradiation, and every day for at least 2 weeks immediately before and after irradiation in the case of single irradiation. Administration is preferred. In particular, when irradiation with heavy particle irradiation is confined to the lesion, it is necessary to administer sufficient NO and NOS inhibitors before and after irradiation in consideration of intense migration of tumor cells.

  In addition, when X-rays and heavy particle beam irradiation are used in combination, X-rays with a safe dose disclosed by conventional technology are used in combination with NO and NOS inhibitors and compared with the range including the tumor mass center and the infiltrated area. It is effective to administer heavy particle beam irradiation after X-ray irradiation, confined to the center of the tumor mass, combined with NO and NOS inhibitors, and administered for more than 2 weeks after irradiation. It is.

  High doses of heavy particle radiation cause brain necrosis, and exposure to heavy particle radiation over a wide range even at low doses may cause serious side effects such as marked brain atrophy and hydrocephalus. It is considered that a safe and effective treatment method is to first perform a wide range of X-ray irradiation, which has been established, and then perform localized irradiation of heavy tumor particles.

  In this case, combined use with NO and NOS inhibitors is necessary, and irradiation alone can only achieve the results of the prior art, and therefore local recurrence, intraspinal dissemination, and invasive growth in the irradiation field may occur. The combined use of NO and NOS inhibitors can be expected to suppress the invasive growth caused by irradiation.

  When a combination of NO and NOS inhibitors is used with a device capable of relatively high dose and split irradiation such as Cyberknife, the therapeutic effect is further improved. Specifically, irradiation is performed in 6 to 8 divisions with a line amount of 5 Gy with a cyber knife device, and the NO and NOS inhibitors are administered for 2 weeks after irradiation. If the lesion is relatively wide and the infiltrated area is large, normal X-ray irradiation is applied to the area including the infiltrated area over a wide range of 40 Gy (2 Gy / day x 20 times), and then the tumor is applied using a cyber knife device. 20Gy (5Gy / times x 4) irradiation is limited to the lump. Also in this case, irradiation alone causes tumor cell migration and increased invasiveness by irradiation, so it is recommended to use NO and NOS inhibitors in combination every day for 2 weeks after irradiation.

  Clinically, in order to reduce side effects of the drug, the drug may be administered only for the first 10 days from the start of divided irradiation. It is also effective in combination with migration inhibitors such as the current standard treatment temozolomide (Temodar (R)), interferon (Feron (R)), and AMPA receptor antagonist (Taranpanel (R)). is there.

  The dosage of the migration inhibitor of the present invention is appropriately determined depending on the individual case in consideration of symptoms, age of the administration subject, sex, etc., but usually 10 to 2000 mg per day for an adult, preferably per day About 150mg. 10-2000 mg per day for an adult may be administered once or divided into 2-4 times. In the case of intravenous administration or continuous intravenous administration, it may be administered at 1 to 24 hours per day. The dose is determined according to the type of active ingredient, the form of migration inhibitor, and the like, but if effective, a dose smaller than the above range can be used.

  The migration inhibitor of the present invention is mainly administered parenterally, specifically subcutaneous administration, intramuscular administration, intravenous administration, transdermal administration, intrathecal administration, epidural, intraarticular, and topical administration. Alternatively, it can be administered in various dosage forms such as oral administration if possible.

  Examples of injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, emulsions and the like. Examples of the aqueous solution and suspension include distilled water for injection and physiological saline. Examples of the water-insoluble solution and suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, polysorbate 80 (trade name), and the like.

  The composition for parenteral administration may further contain adjuvants such as preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizing agents (eg lactose), solubilizing agents (eg megluminic acid). . These are sterilized by, for example, filtration through a bacteria-retaining filter, blending of bactericides, or light irradiation. A composition for parenteral administration can be prepared by preparing a sterile solid composition and dissolving it in sterile water or a sterile solvent for injection before use.

  When the migration inhibitor of the present invention is used as a solid composition for oral administration, it can be in the form of tablets, pills, powders, granules and the like. Such a solid composition includes, for example, an inert diluent such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, metasilicic acid, magnesium aluminate, and the active substance as an active ingredient. And can be prepared by mixing.

  For solid compositions, additives other than inert diluents, for example, lubricants such as magnesium stearate, disintegrants such as calcium calcium glycolate, stabilizers such as lactose, glutamic acid and aspartic acid are used in accordance with conventional methods. A solubilizing agent such as can be blended. Tablets and pills may be coated with sugar coating such as gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate or the like, or a film of gastric or enteric substance, if necessary.

  When the migration inhibitor of the present invention is used as a liquid composition for oral administration, it contains a pharmacologically acceptable emulsion, suspension, syrup, elixir, etc. Contains agents such as purified water and ethanol. In addition to the inert diluent, the composition may contain adjuvants such as wetting agents and suspending agents, sweeteners, flavors, fragrances and preservatives.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples at all.

In the following embodiments, the following means are employed.
1) Surgical specimen and cell culture The surgical specimen examined in this example was histologically identified as a polymorphic glioblastoma cell according to the classification of the World Health Organization. Cell cultures were prepared according to previous reports. CGNH-PM, a subline of CGNH-89, was also used. Cell culture was performed in Eagle's minimal essential medium (Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum and 2 mM glutamine.
2) X-ray irradiation As for the irradiation of cells, the MBR-1505R X-ray device (manufactured by Hitachi) operating at 140kV and 4.5mA is used with 0.5mm Al filtration, and the focal source distance is 30cm and 1.11Gy / min. Akimoto, T. et al. Int. J. Radiat. Oncol. Bial. Phys. 50, 195-201 (2001)).
3) Carbon beam irradiation Carbon beam irradiation was performed using a ¹² C ion beam (220 MeV), LET; linear energy transfer, 108 KeV / μm broad beam made by AVF cyclotron, Japan Atomic Energy Agency. . During the irradiation, the culture supernatant was removed and the tumor cells were covered with a polyimide film (Kapton; DuPont-Toray Co., Ltd.) having a thickness of 8 μm to prevent drying.
4) Immunofluorescence Indirect fluorescent antibody staining was performed according to the previous report (Huang, X. et al. Proc. Natl. Acad. Sci. USA 102, 1065-1070 (2005)). For double immunofluorescence, the bound antibody was visualized using fluorescein isothiocyanate and a rhodamine-conjugated secondary antibody (Molecular Probes, Inc.). Stained cells were examined using a laser scanning confocal microscope (Pascal LSM5; Carl Zeiss). Deoxyribonucleic acid counterstaining was performed using DAPI. Primary antibodies used were Phospho-Akt (Ser-473; Cell Signaling Technology), vimentin (V9; Dako), GluR1 (C-terminal) (Chemicon), GluR1 (N-terminal) (Carbiochem), GluR1 (S845 phosphorrylated) ( Upstate), cGKII (Santa Cruz Biotechnology, Santa Cruz, CA), iNOS (BD Transduction Laboratories), GFAP (DAKO).
5) Migration assay In the migration assay, a glass cloning cylinder (diameter 7 mm) coated at one end with silicon grease was placed in the center of the culture dish. 5 × 10 ⁴ cells were seeded in this cylinder. At 24 hours after the plating, the cells were irradiated with radiation to remove this cloning ring. Cells were cultured for an additional 48 hours. Subsequently, the number of cells exceeding the boundary of this cloning circle was counted.
6) Identification and labeling of NO-producing cells
Identification and labeling of NO-producing cells were performed using diaminofluorescein-2 diacetate (DAF-2 DA, used at 10 μM, Daiichi Chemicals Co., Ltd.) (Nakatsubo, Kojima FFBS Letters 427,273-266, 1998). The distribution of NO-producing cells was captured by visualizing DAF-2T fluorescence converted from DAF-2 in the presence of NO with a confocal microscope (Pascal LSM5; Carl Zeiss).
7) Surface GluR1 cross linking.
The collected cells were cooled with ice-cold PBS and stirred with 2 mM BS3 (Pierce, Rockford, IL) for 30 min at 4 ° C. Half was filled with BS3 and stirred only with ice-cold PBS. Quench with 100 mM glycine (10 min, 4 ° C) to complete cross-linking. Pellets were lysis buffers (25 mM HEPES, pH 7.4, 500 mM NaCl, 2 mM EDTA, 1 mM DTT, 1 mM phenylmethyl sulfonyl fluoride, 20 mM NaF, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM microcystin-LF, Suspended with 1 μM okadaic acid, 1 μL protease inhibitor mixture (Calbiochem, La Jolla, CA), and 0.1% Nonidet P-40 (v / v)], rotated for 5 seconds, homogenized, and Lowry method (Lowry et al. , 1951).
8) Data analysis Data are shown as mean ± standard error. Statistical comparisons were performed using unpaired t-tests or one-way analysis of variance (Schaffe test for post hoc analysis).
<Example 1>
The molecular mechanism of the enhancement of radiation-induced invasive growth via nitric oxide was analyzed from migration experiments using clinical specimens.

  In the migration ability evaluation test of FIG. 2, it was confirmed by X-ray and carbon beam irradiation that the migrating cells exhibited expression of NO immediately after irradiation, and this NO was subsequently transmitted to non-irradiated cells. Further, as shown in FIG. 3, it was found that the mobility of the cells is lowered by the NO neutralizer (C-PTIO).

  As shown in FIG. 4, NO synthesis inhibitor L-NAME also reduces cell mobility, and NOS1 (nNOS), NOS2 (iNOS), NOS3 (eNOS) selectively interferes with RNA and NOS2 alone Inhibition of NOS2 (iNOS) expressed in tumor cells was confirmed to be important for suppressing NO production.

  RNA interference experiments for NOS were performed using RNAi probes for NOS1, NOS2, and NOS3 purchased from Invitrogen. The probe sequence used in the RNA interference experiment is as follows.

  Fluorescent Oligo (Invitrogen) was co-transfected with the probe as an introduction marker.

  The present inventor has reported that phosphorylation of Ser473 of Akt is induced by intracellular influx of calcium via a calcium permeable AMPA receptor, and this leads to enhanced cell migration (J Neuroscience 27: 7987-8001,2007). ). It was shown in Patent Document 3 that phosphorylation of Ser473 of Akt occurs at the leading edge of migration of tumor mass under irradiation. In FIG. 5, it was considered that Akt is a substrate for NO because tumor interference with RNA in advance and knockout of Akt not only inhibits cell migration but also reduces NO production. It was revealed that NO-AMPA receptor-Akt signaling is involved in the background of the molecular mechanism of cell migration promoted by irradiation. In the RNA interference experiment of Akt, transfection was performed using an RNAi probe for Akt1 purchased from Invitrogen. The probe sequence used is as follows.

  Fluorescent Oligo (Invitrogen) was co-transfected with the probe as an introduction marker.

  As shown in Fig. 6, from the calcium photometry experiment using Fluo-3, spindle cells with enhanced migration showed about twice the increase in intracellular calcium by AMPA + CTZ stimulation compared to non-irradiated cells. I confirmed that it was rising.

From the above, radiation promotes NO production via NOS2, doubles the increase of intracellular calcium via calcium-permeable AMPA-type receptor expressed in tumor cells, and promotes phosphorylation of serine 473 of Akt. It was found that increased migration was induced. Akt was also found to be a substrate for NO. From these, the conceptual diagram of FIG. 1 was created.
<Example 2>
The molecular mechanism of GluR1 subunit translocation mechanism was analyzed.

  In FIG. 7, a cell surface receptor is identified by live-staining the cell using an antibody that recognizes the extracellular domain N-terminus of GluR1, and the expression level thereof is analyzed. As in the case of radiation treatment, NOC-18 and 8-Br-cGMP treatment groups, which are NO donors, doubled expression in the cell membrane of GluR1. No increase in the expression level was observed in the group in which NO-C-18 was simultaneously administered with NO-neutralizing agent C-PTIO, and in the group in which 8-Br-cGMP was administered with KT5823, a selective inhibitor of cGMP dependent kinase II (cGKII). . Increased expression is suppressed when KT5823 is used in combination with irradiation, expression is doubled even when cPTIO is co-administered with 8Br-cGMP, and expression is observed when 8Br-cGMP is used concomitantly with C-PTIO for irradiation The doubling of cGKII suggests that cGKII intervention is essential for the movement of the GluR1 subunit into the cell membrane.

  FIG. 8 is a double-stained image of the GluR1 antibody and cGKII antibody that recognizes the N-terminal, and the non-irradiated and irradiated groups were consistent with surface GluR1 and the localization of cGKII was observed.

From the above, it was found that cGKII is involved in the movement of the GluR1 subunit to the cell membrane.
<Example 3>
Analysis of cell membrane components and cytoplasmic components of GluR1 by crosslinking assay As shown in FIG. 9, the receptor on the membrane surface is doubled by radiation compared to non-irradiated cells. The ratio of cytoplasmic and cytoplasmic components of GluR1 is also half for cytoplasmic and membrane components in non-irradiated cells, but in irradiated cells, 3/4 of all receptors are membrane components 1/4 of cytoplasmic components. found. Thus, NO was found to be involved in the redistribution of calcium-permeable AMPA-type receptors in tumor cells.
<Example 4>
Induction of Ser845 phosphorylation of GluR1 by irradiation In FIG. 10, phosphorylation of Ser845 of GluR1 is induced by irradiation, and this phosphorylation promotes the transfer of GluR1 to the cell membrane. Both X-rays and carbon rays promote phosphorylation of GluR1 about 1.5 times compared to the non-irradiated control. The NO neutralizer, cPTIO, suppresses the promotion of phosphorylation, and therefore has found the importance of NO in phosphorylation of Ser845 of GluR1.
<Example 5>
About the importance of NO-GluR1 signaling Figure 11 shows the bystander effect of NO signal in enhancing the migration of tumor cells by irradiation, and shows that GluR1 is important for the propagation of this signal to non-irradiated cells Yes.
<Example 6>
Identification of iNOS-NO-cGKII-GluR1 signaling using clinical specimens immediately after radiotherapy (within 1 month).

Immunohistological analysis of iNOS, cGKII, and GluR1 protein expression was performed using 11 surgically removed clinical specimens. As controls, 14 specimens of non-irradiated tissue were used.
Extracting findings characteristic of irradiated tissue,
1) Extensive necrotic tissue. Ischemic necrosis with occlusion of blood vessels
2) Vitrification and occlusion of tumor blood vessels 3) Invasive proliferation of circular small cells or spindle cells around wide nests and presence of new blood vessels 4) Neurons such as the appearance of a small number of splintered elephants and bizarre polygonal giant cells Presents with pathological findings.

  As shown in FIG. 11, significant iNOS, cGKII, and GluR1 protein expression was observed at the most advanced site of invasive growth of irradiated tissues. Such a finding was not observed in the so-called necrosis pseudopalisading region of the non-irradiated tissue.

  As described above, iNOS-NO-cGKII-GluR1 signaling was identified by Examples 1 to 6 and confirmed by clinical samples. As shown in the conceptual diagram of Fig. 1, when using radiotherapy, it is important to block the NO and NOS-mediated signals of increased migration, and here we prove the usefulness of the combined use of NO and NOS inhibitors. did it.

Claims (1)

It is a migration inhibitor that inhibits the enhancement of tumor cell migration associated with irradiation of tumor cells in radiation cancer treatment, and has the activity of inhibiting the production of nitric oxide (NO) and nitric oxide synthase (NOS) Having the following compounds (1) (2),
(1) 2- (4-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO),
(2) NG-Nitro-L-arginine Methyl Ester, Hydrochloride (L-NAME),
Yu run inhibitors you characterized by containing at least one of.