Patent Application
20050232915
This invention relates to organic compounds, in particular to pharmaceutical compositions for use in combination with cytotoxic therapy and heat shock for the treatment of tumors.
Carcinoma is by far the most common type of cancer; it accounts for about 80% of all cases of cancer. The severity of a carcinoma can vary widely with pancreatic cancer being one of the most aggressive and lethal neoplasms with an extremely low 5-year survival rate; Landis, S. et al (CA Cancer J. Clin., 49: 8-31, 1999) and Niederhuber, J. E. et al (Cancer, 76:1671-1677, 1995). Because most patients with pancreatic cancer miss the opportunity for complete surgical resection at the time of diagnosis, radiotherapy remains as a major component of treatment modalities for controlling tumor progression. Malignant progression of pancreatic cancer depends not only on rapid proliferation of tumor cells but also on other biological behaviours including motility, invasiveness, and metastatic potential. More generally radiotherapy remains a major therapeutic option for patients with various other types of advanced cancer. Radiotherapy besides having the desired effect also has an effect on malignant biological behaviours for example it has now been found that while it significantly inhibits cell proliferation and migration irradiation may enhance the invasive potential in pancreatic cancer cells.
Current treatments for cancer are effective to some extent but all have some undesirable effects and carry risks which need to be taken into account when choosing a specific treatment. The side effects of some treatments also include the promotion of the cancer. A treatment that has all the benefits of the current treatments but without or with a reduced risk of promoting the development of the cancer would be highly beneficial.
We have now found that certain matrix metalloproteinase inhibitors are effective when used in combination with radiotherapy therapy for the treatment of tumors especially tumors of the brain, breast, larynx, pancreas, skin, tongue, uterine cervix also leukaemia and lymphoma. Further we have found that such matrix metalloproteinase inhibitors may be used in combination with heat shock in combination with additional cytotoxic therapy for the treatment of such tumours.
Accordingly in a first aspect the invention provides a method of treating cancer in a subject in need of such treatment which comprises administering to the subject an effective amount of a matrix metalloproteinase inhibitor in combination with radiotherapy.
Accordingly in a second aspect the invention provides a method of treating tumors in a subject in need of such treatment which comprises administering to the subject an effective amount of a matrix metalloproteinase inhibitor in combination with heat shock and cytotoxic therapy
Preferably the invention provides a method of treating tumors in a subject in need of such treatment which comprises administering to the subject an effective amount of a hydroxamic acid derivative matrix metalloproteinase inhibitor (of the formula I) in combination with
Hydroxamic acid derivative metalloproteinase inhibitors are well known in the art. A suitable metalloproteniase inhibitor for use in the method of the invention is, for instance, a compound of formula I
(i) Wherein
Further the invention provides the use of a hydroxamic acid derivative metalloproteinase inhibitor, for instance a compound of formula I (or pharmaceutically acceptable salt or prodrug ester thereof) for the preparation of a medicament for use in combination with
In a further aspect the invention provides use of a hydroxamic acid derivative metalloproteinase inhibitor, for instance a compound of formula I (or pharmaceutically acceptable salt or prodrug ester thereof) in combination with
In yet further aspect the invention provides a hydroxamic acid derivative matrix metalloproteinase inhibiting agent comprising, for instance a compound of formula I (or pharmaceutically acceptable salt or prodrug ester thereof) as active ingredient for use in combination with
In a yet further aspect the invention provides a package comprising a hydroxamic acid derivative metalloproteinase inhibitor, for instance a compound of formula I (or pharmaceutically acceptable salt or prodrug ester thereof) together with instructions for the use in combination with
The invention may be used for the treatment of any tumor which is susceptible to treatment by cytotoxic therapy, including the treatment of solid tumours, carcinoma, adenocarcinoma. For example the invention may be used in the treatment of tumors of the brain, breast, larynx, skin, tongue, uterine cervix and also leukaemia and lymphoma, especially pancreatic tumors.
Above and elsewhere in the present description the following terms have the meanings given below:
The term “lower” referred to above and hereinafter in connection with organic radicals or compounds respectively defines a compound or radical which may be branched or unbranched with up to and including 7, preferably up to and including 4 carbon atoms.
A lower alkyl group is branched or unbranched and contains 1 to 7 carbon atoms, preferably 1-4 carbon atoms. Lower alkyl represents, for example, methyl, ethyl, propyl, butyl, isopropyl or isobutyl.
A lower alkoxy (or alkyloxy) group preferably contains 1-7 carbon atoms, advantageously 1-6 carbon atoms, and represents for example methoxy, ethoxy, propoxy, isopropoxy, isobutoxy, preferably methoxy. Lower alkoxy includes cycloalkyloxy and cycloalkyl-lower alkyloxy.
Halogen (halo) preferably represents chloro or fluoro but may also be bromo or iodo.
Aryl represents carbocyclic or heterocyclic aryl including biaryl.
Carbocyclic aryl represents monocyclic, bicyclic or tricyclic aryl, for example phenyl or phenyl mono-, di- or tri-substituted by one, two or three radicals selected from lower alkyl, lower alkoxy, hydroxy, halogen, cyano, trifluoromethyl, lower alkylenedioxy, and oxy-C2-C3-alkylene; or 1- or 2-naphthyl. Lower lkylene is a divalent substituent attached to two adjacent carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent attached to two adjacent carbon atoms pf phenyl, e.g. oxyethylene or oxypropylene, An example for oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.
Heterocyclic aryl represents monocyclic or bicyclic heteroaryl, for example pyridyl, indolyl, quinoxalinyl, quinolyl, isoquinolyl, benzothienyl, benzofuranyl, benzopyranyl, benzothiopyranyl, furanyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any said radical substituted, especially mono- or di-substituted, by lower alkyl or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl, advantageously 2-thienyl. Quinolyl represents 2-, 3- or 4-quinolyl, advantageously 2-quinolyl. Isoquinolyl represents preferably 1-, 3- or 4-isoquinolyl. Benzopyranyl, benzothiopyranyl represent preferably 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl represents preferably 2- or 4-thiazolyl, advantageously 4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl. Imidazolyl is preferably 4-imidazolyl.
Biaryl is preferably carbocyclic biaryl, e.g biphenyl, namely 2, 3 or 4-biphenyl, advantageously 4-biphenyl, each optionally substituted by e.g. lower alkyl, lower alkoxy, halogen, trifluoromethyl or cyano.
Cycloalkyl represents a saturated cyclic hydrocarbon optionally substituted by lower alkyl which contains 3 to 8 ring carbons and is advantageously cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl optionally substituted as hereinbefore defined; cycloalkyl includes heterocyclyl.
Heterocyclyl represents a saturated cyclic hydrocarbon containing one or more, preferably 1 or 2, hetero atoms selected from O, N or S, and preferably from 3 to 10, more preferably 5 to 8, ring atoms; for example, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyrrolyl, piperidinyl, piperazinyl or morpholino; all of which may be optionally substituted, for instance as hereinbefore defined.
Amino may be optionally substituted, e.g. by lower alkyl.
Aryl-lower alkyl represents preferably (carbocyclic aryl or heterocylic aryl)-lower alkyl.
Carbocyclic aryl-lower alkyl preferably represents aryl-straight chain or -branched C1-4-alkyl in which carbocyclic aryl has meaning as defined above, e.g. benzyl or phenyl-(ethyl, propyl or butyl), each unsubstituted or substituted preferably on the phenyl ring as hereinbefore defined for carbocyclic aryl above.
Heterocyclic aryl-lower alkyl represents preferably straight chain or branched heterocyclic aryl-C1-7-alkyl in which heterocyclic aryl has meaning as defined above.
Cycloalkyl-lower alkyl represents e.g. (cyclopropyl- or cyclobutyl)-(methyl or ethyl).
Combination refers to all combinations, of a MMP inhibitor of formula I,
The radiotherapy or heat shock and cytotoxic therapy can be continuous, sequential or sporadic. Preferably the effect obtained is such as would not be obtained if there is cytotoxic therapy without prior, simultaneous or subsequent radiotherapy or heat shock therapy with administration of a MMP inhibitor of formula I. Radiotherapy, heat shock or administration of MMP inhibitor of formula I may be continuous, sequential or sporadic
Preferably combination refers to all combinations, of a MMP inhibitor of formula I, radiotherapy or heat shock and cytotoxic therapy, such that there is an effect on MMP expression or tumour invasion potential which would not be obtained if
Thus in a particularly preferred embodiment the invention provides a method of treating cancer in a subject in need of such treatment which comprises administering to the subject an effective amount of a matrix metalloproteinase inhibitor in combination with radiotherapy and heat shock treatment.
Heat shock refers to any method of causing a heat shock response by a cell or cells in a tumor or within the area of a tumor. Heat shock may be administered to the whole body, part of the body or locally to the tumor and may be caused by external or internal means, for example heating rods, microwaves, radiofrequences, ultrasound, thermal blankets, thermal baths, lasers, inducing fever e.g administration of a pyrogen, etc.
Radiotherapy may may comprise any of the forms of radiation therapy used or proposed for use in treatment of cancers, including tumours. Thus, for example, gamma radiation may be used or X-ray radiation or any of the other forms of radiation customarily used for cancer treatment.
The term “tumor” is intended to mean malignant tumors and benign tumors in particular cancerous tumors for example cancers of the brain, breast, larynx, pancreas, skin, tongue, uterine cervix also leukaemia and lymphoma.
Preferred embodiments provide a method of treating tumor which can be treated with cytotoxic therapy in a subject in need of such treatment which comprises cytotoxic therapy and heat shock in combination with administering to the subject an effective amount of;
Particularly preferred embodiments provide a method of treating cancer which can be treated with radiotherapy in a subject in need of such treatment which comprises radiotherapy and/or heat shock in combination with administering to the subject an effective amount of;
The configuration of the Compounds of formula VI′ is preferably that of Formula VIa:
or of Formula VIb:
most preferably that of Formula VIa.
Further particularly preferred embodiments provide a method of treating cancer which can be treated with radiotherapy in a subject in need of such treatment which comprises radiotherapy and/or heat shock in combination with administering to the subject an effective amount of:
Further and most preferred embodiments provide a method of treating a tumour in a subject in need of such treatment which comprises administering to the subject an effective amount of a pharmaceutical composition for use in combination with heat shock and/or radiotherapy wherein said pharmaceutical composition comprises
Compounds of formula I, II, III, IV, V, VI an VII and their synthesis are described in published international patent applications Nos. WO 98/14424, WO 97/22587 and EP 606046, the teachings of which are incorporated herein by reference.
The agents of the invention, i.e. the MMP inhibitors of formula I and pharmaceutically acceptable salts and prodrug derivatives, are preferably used in the form of pharmaceutical preparations that contain the relevant therapeutically effective amount of active ingredient optionally together with or in admixture with inorganic or organic, solid or liquid, pharmaceutically acceptable carriers which are suitable for administration.
The MMP inhibitor pharmaceutical compositions may be, for example, compositions for enteral, such as oral, rectal, aerosol inhalation or nasal administration, compositions for parenteral, such as intravenous or subcutaneous administration, or compositions for transdermal administration (e.g. passive or iontophoretic), or compositions for topical administration,
Preferably, the MMP inhibitor pharmaceutical compositions are adapted to oral administration.
The particular mode of administration and the dosage may be selected by the attending physician taking into account the particulars of the patient, especially age, weight, life style, activity level, etc.
The dosage of the Agents of the invention may depend on various factors, such as effectiveness and duration of action of the active ingredient, mode of administration, and/or sex, age, weight and individual condition of the subject to be treated.
The agents of the invention are useful in the manufacture of pharmaceutical compositions comprising an effective amount thereof in conjunction or admixture with excipients or carriers suitable for either enteral or parenteral application. In addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.
Parenteral formulations are especially injectable fluids that are effective in various manners, such as intravenously, intramuscularly, intraperitoneally, intranasally, intradermally or subcutaneously. Such fluids are preferably aqueous isotonic solutions or suspensions that can be prepared before use, for example from lyophilised preparations that contain the active ingredient alone or together with a pharmaceutically acceptable carrier. The pharmaceutical preparations be sterilised and/or contain adjuncts, for example preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers.
Suitable oral forms are tablets and gelatin capsules comprising the active ingredient together with a) diluents, e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g. silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders e.g. magnesium aluminium silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g. starches, agar, alginic acid, or it's sodium salt, or effervescent mixtures; and/or e) adsorbents, colorants, flavours and sweeteners. Tablets may be either film coated or enteric coated according to methods known in the art.
Suitable formulations for transdermal application include an effective amount of a compound of the invention with carrier. Advantageous carriers include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
Suitable formulations for topical application, e.g. to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels, or sprayable formulations, for example, for delivery by aerosol or the like. Such topical formulations typically contain from about 0.1 up to about 50% by weight, preferably from about 1 up to about 20% by weight, of MMP inhibitor.
The following examples are intended to illustrate the invention and are not to be construed as being limitations thereon.
Tablets each containing 50 mg of N-hydroxy-2 (R)-[[4-methoxyphenylsulfonyl](3-picolyl)-amino]-3-methylbutanamide hydrochloride can be prepared as follows:
The active ingredient is mixed with the lactose and 292 g of potato starch, and the mixture is moistened with an ethanolic solution of the gelatin and granulated through a sieve. After the granules have dried, the remainder of the potato starch, the magnesium stearate and the silicon dioxide are admixed and the mixture compressed to give tablets each weighing 145.0 mg and containing 50.0 mg of active ingredient, which can, if desired, be provided with breaking grooves to enable the dosage to be more freely adjusted.
Preparation of 3000 capsules each containing 25 mg of the active ingredient, for example, N-hydroxy-2 (R)-[[4-methoxyphenylsulfonyl](3-picolyl)-amino]-3-methylbutanamide hydrochloride:
The active ingredient is passed through a No. 30 hand screen.
The active ingredient, lactose, Avicel PH 102 and Polyplasdone XL are blended for 15 minutes in a mixer. The blend is granulated with sufficient water (about 500 mL), dried in an oven at 35° C. overnight, and passed through a No. 20 screen.
Magnesium stearate is padded through a No. 20 screen, added to the granulation mixture, and the mixture is blended for 5 minutes in a mixer. The blend is encapsulated in No. 0 hard gelatin capsules each containing and amount of the blend equivalent to 25 mg of the active ingredient.
Materials and Methods
Cell Culture and Reagents
Three human pancreatic cancer cell lines are used in this study. Panc-1 and Suit-2 are generously provided by Dr. Iguchi (National Kyushu Cancer Center, Fukuoka, Japan), Hs766T is obtained from American type culture collection (Rockville, Md.). Cells are maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma Chemical Co. St. Louis, Mo., USA) supplemented with 10% fetal bovine serum (FBS), streptomycin (100 μg/ml), and penicillin (100 U/ml) at 37° C. with humidified 90% air and 10% CO2. The number of cells is counted with a particle distribution counter, CDA500 (Sysmex, Kobe, Japan). The MMP inhibitor N-hydroxy-2(R)-[[4-methoxyphenylsulfonyl](3-picolyl)amino]-3-methyl-butaneamide hydrochloride)monohydrate, is kindly provided by Novartis Pharma, K.K., Japan.
Irradiation
The cells are irradiated with doses of 3, 5, or 10 Gy at room temperature using a 137Cs source (Gamma Cell 40, Atomic Energy of Canada Ltd., Ontario, Canada) delivering 1.0 Gy/min.
Cell Proliferation Assay
Cell proliferation is evaluated by measuring the fluorescence intensity of propidium iodide (PI) as described previously by Zhang et al. (Cancer Lett., 142: 129-137, 1999) with minor modifications. Briefly, cells are seeded in 24-well plates at a density of 3×104 cells per well. After overnight cultivation, cells are irradiated and cultured for 4 days. PI (30 μM) and digitonin (600 μl) are added to each well to label all nuclei of the cells with PI. Fluorescence intensity corresponding to total cells in each well is measured by a multi-well plate-reader, CYTOFLUOR II (PerSeptive Biosystems Inc., Framingham, Mass., USA) with 530-nm excitation and 645-nm emission filters. The cell proliferation rate is calculated as the proportion of fluorescence intensity of each well at the time point indicated in the text to that at the day of irradiation.
Migration Assay
Migration of pancreatic cancer cells through 8 μM pores is assessed using the Transwell cell culture chamber (6.5 mm diameter, Corning Costar, Tokyo, Japan) as described by sato et al and Maehara et al (Cancer, 91: 496-504, 2001; Br. J. Cancer, 84: 864-873, 2001). Cells at a density of 1×104 are seeded in the upper chambers with 100 μl of medium supplemented with 10% FBS. Same media of 600 μl are placed in the lower wells. After seeding, the cells are subjected to irradiation and then cultured for 24 h. The filter membranes are removed and fixed with 70% ethanol and stained with hematoxylin and eosin (H&E). The number of cells that had migrated to the lower surface of the filter membrane is counted in five random fields under a light microscope.
Matrigel Invasion Assay
Invasion of pancreatic cancer cells is measured by the invasion of cells through Matrigel-coated transwell inserts (Becton Dickinson, Franklin Lakes, N.J., USA) (Sato et al and Maehara et al ibid).
Briefly, transwell inserts with 8 μm pore are coated with Matrigel (40 μg/well, Becton Dickinson, Bedford, Mass., USA). Five hundred μl of cell suspension (1×105/ml) are added to the upper chambers. Same media of 750 μl are placed in the lower wells. Thereafter, the cells are irradiated and incubated for 24 h. Cells that have invaded to lower surface of the Matrigel-coated membrane are fixed with 70% ethanol, stained with H&E, and counted in five random fields under a light microscope.
Gelatin Zymography
The conditioned medium either from non-irradiated or irradiated Panc-1 cells is concentrated to 10-fold with Centricon-10 (Amico, Beverly, Mass., USA). Samples are added to each lane and subjected to 10% SDS-polyacrylamide gel electrophoresis, using 10% polyacrylamide gel containing 1 mg/ml gelatin. After electrophoresis, the gel is washed in 2.5% Triton X-100, and incubated in 50 mM Tris-HCl buffer (pH8.0) containing 0.5 mM CaCl2 and 1 mM ZnCl2 for 20 hr at 37° C. The gel is stained with 1% Coomassie Brilliant Blue R-250 and destained with destaining buffer (5% acetic acid and 10% methanol).
Western Blotting
The proteins (80 μg/lane) from the soluble fraction of Panc-1 cells are fractionated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, Mass.). The membrane is incubated with 1:500 dilutions of polyclonal antibody for human uPA (urokinase-type plasminogen activator, Santa Cruz Biotechnology, Calif., USA), and then probed with anti-goat IgG conjugated with horseradish peroxides (Santa Cruz Biotechnology, Calif., USA). Immunoblots are detected by the enhanced chemiluminescence (Amersham International, Buckinghamshire, UK).
Statistical Analysis
Statistical analyses are performed by using ANOVA and unpaired Student's t test. All statistics are performed on two-sided test. P<0.05 is considered as significant. Each experiment is repeated at least three times.
Results
Irradiation Inhibits Proliferation of Pancreatic Cancer Cells
First, we examine the proliferation of pancreatic cancer cells after irradiation. Irradiation suppressed the proliferation of Panc-1 cells in a dose-dependent manner, and an almost complete inhibition is observed at a dose of 10 Gy. Similar results are obtained in Suit-2 at the same dose range. In Hs766T cells, however, while dose reached to 5 Gy, radiation had already entirely inhibited the cell growth.
Irradiation Promotes Invasive Potential but Inhibits Migration Ability in a Subset of Pancreatic Cancer Cells
To determine the effect of radiation on cell motility, we analyse the migration of human pancreatic cancer cells before and after irradiation using the Transwell cell migration assay. Compared with untreated controls, Panc-1 and Suit-2 cells irradiated at doses of 3, 5, and 10 Gy show significantly lower numbers of migrated cells. There is no significant change in migration potential after irradiation in Hs766T cells, which show a relatively low basal migration activity.
We next examine changes in the invasive potentials of pancreatic cancer cells after irradiation using the Matrigel invasion assay. In contrast to the decline in migration ability, invasive potentials in both Panc-1 and Suit-2 cells are significantly increased after irradiation at doses of 3, 5, and 10 Gy. This increase in invasive potential appears to be dose-dependent. Remarkably, the average number of invaded cells in Panc-1 is increased by more than 2-fold after irradiation at 10 Gy. We find no significant change in invasive potential in irradiated Hs766T cells.
Irradiation Increases MMP-2 Activity
To determine the role of gelatinases in the radiation-induced changes in invasive potential, we examine MMPs activity in Panc-1 cells before and after irradiation. Cells are incubated 24 h after irradiation and the conditioned medium is subjected to the gelatin zymography. Untreated Panc-1 cells secrete both latent and active forms of MMP-2 (72 kDa and 62 kDa gelatinases). After irradiation, MMP-2 activity of either latent or activated type is significantly increased, thus suggesting that the increased MMP-2 activity may play an important role in the enhanced invasiveness after irradiation.
An MMP Inhibitor Blocks the Radiation-Enhanced Invasion of Pancreatic Cancer Cells
Finally, we test whether a synthetic MMP inhibitor, N-hydroxy-2(R)-[[4 methoxyphenylsulfonyl](3-picolyl)amino]-3-methyl-butaneamide hydrochloride) monohydrate, could prevent the radiation-enhanced invasiveness. N-hydroxy-2(R)-[[4-methoxyphenylsulfonyl](3-picolyl)amino]-3-methyl-butaneamide hydrochloride) monohydrate is added to invasion chambers at final concentrations of 1, 5, and 10 μm just before irradiation. After irradiation at 5 Gy, the number of invaded cells in Panc-1 increase from 14.6 cells/field to 24.4 cells/field, whereas concomitant treatment with N-hydroxy-2(R)-[[4-methoxyphenylsulfonyl](3-picolyl)amino]-3-methyl-butaneamide hydrochloride)monohydrate at concentrations of 5 and 10 μM significantly block the increase in invaded cells after irradiation. Treatment with N-hydroxy-2(R)-[[4-methoxyphenylsulfonyl](3-picolyl)amino]-3-methyl-butaneamide hydrochloride) monohydrate does not affect the growth and viability of Panc-1 cells at concentrations up to 10 μM. Furthermore, gelatin zymography reveals that treatment with N-hydroxy-2(R)-[[4-methoxyphenylsulfonyl](3-picolyl)amino]-3-methyl-butaneamide hydrochloride) monohydrate at 5 μM markedly decreases the active type MMP-2 without affecting the enzymatic activity of latent type MMP-2.
Expression of Urokinase-Type Plasminogen Activator (uPA) Decreases After Irradiation
To determine the possible involvement of uPA in the changes in cell motility after irradiation, we examine the expression of uPA in Panc-1 cells by Western blotting. The uPA expression in cell lysate that represents the constituent portion of uPA is suppressed by irradiation.
Acquisition of Array Data
For gene expression analysis, HeLa cells seeded in 100 mm petri dish are dipped in a water bath for 1 h at 44° C. (±0.03° C.). RNA is isolated from cells 0, 3, 6 and 12 h after heating. The cells just before heat shock treatment are used as control. Labeled probe is hybridized to a Human 1 cDNA microarray (no. G4100A; Agilent Technologies). The gene expression experiment is repeated two times.
Data analysis
Signal intensities of Cy3 and Cy5 from the 12,814 spots are quantified and analyzed by GenePix (Axon Instruments, Foster City, Calif.). Previously fagged spots by GenePix and 60% of spot pixels with intensities more than one standard deviation above the background pixel intensity are excluded. Residual spot signals are normalized so that median of all signal ratio (Cy3/Cy5) is 1.0. Then extract the genes that showed Cy3/Cy5 signal ratio >2.0 or 0.5<at both two times experiment.
Results
Analysis of Gene Expression Profiles from Data Preprocessing
752 genes are up or down-regulated after heat shock. The temporal pattern of expression for 752 genes is more easily recognized through clustering. Using Fuzzy ART, those genes are separated into 8 clusters. Up-regulated genes at 0 h play an important role in repair of injured cells. “Cluster 1” and “Cluster 2”, containing 53 genes are selected and “Cluster 2” included HSP70 which is well known as heat shock response gene. Among these genes, focus on Matrix metalloproteinase 3 (MMP-3), which is included in “Cluster 2” and conduct next experiment.
Inhibitory Effect Using MMP-3 Inhibitor
MMP-3 inhibitor (no. 444225; CALBIOCHEM) is dissolved in DMSO. The final concentration of MMP-3 Inhibitor in each culture medium is 13 μM. With the same concentration DMSO is used as control. MMP-3 inhibitor is added 1 h before heat shock and dishes are dipped in water bath at 44° C. for 60, 75 and 90 min to make the surviving curve. Surviving cells are counted by trypan blue dye exclusion method after 3 days. MMP-3 Inhibitor induced much more cell death than DMSO. These data indicates that MMP-3 appears to play an important role in restoration of injured cells, and thus inhibition of MMP-3 should inhibit recovery of injured cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0205537.4 | Mar 2002 | GB | national |
| 0229054.2 | Dec 2002 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/02365 | 3/7/2003 | WO | 6/6/2005 |
