Patent Application
20060228347
1. Field of the Invention
The present invention relates to an antitumor agent and a novel DNase.
2. Description of the Related Art
A remarkable feature of cancer cells is an unregulated proliferation potency. A group of antitumor agents target a DNA, and it has been clarified that the damaged DNA activates a specific apoptotic pathway. Apoptosis is not a necrosis, which is a mere cell lysis, but an active cell death regulated by genes (British Journal of Cancer, 1972, Vol. 26, p. 239).
As such an antitumor agent which targets a DNA and damages the DNA, for example, an alkylating agent, a topoisomerase inhibitor, and Ara-C (cytosine arabinoside) are known. The alkylating agent causes an irreversible DNA cleavage by alkylating a base portion of a DNA (adduct formation). The topoisomerase inhibitor causes a similar DNA cleavage by stabilizing an intermediate complex (cleavable complex) of a topoisomerase and a DNA. Ara-C is mistaken as deoxyadenosine (a material for a DNA) and incorporated into a DNA, to damage the DNA by inhibiting a DNA polymerase.
When a DNA is damaged by the antitumor agent, apoptosis is finally caused by a DNase, and cancer cells are excluded.
However, that a DNase per se is used as an antitumor agent, and that a DNase does not act on normal cells but specifically acts on cancer cells, is unknown. Furthermore, although a restriction enzyme is known to digest a DNA at a specific recognition site, that a restriction enzyme per se is used as an antitumor agent, and that a restriction enzyme does not act on normal cells but specifically acts on cancer cells, is unknown.
An object of the present invention is to provide an antitumor agent which does not act on normal cells, but specifically acts on cancer cells, i.e., an antitumor agent having few adverse effects, and a novel DNase useful as an active ingredient therefor.
The present invention relates to an antitumor agent comprising as an active ingredient a DNase.
The present invention relates to a method for treating or preventing cancer, comprising administering to a subject in need thereof a DNase in an amount effective in treating or preventing cancer.
The present invention relates to the use of a DNase in the manufacture of an antitumor agent.
According to a preferred embodiment of the present invention, the DNase is used in the form of a complex of the DNase and a liposome.
According to another preferred embodiment of the present invention, the DNase is a restriction enzyme or a novel DNase, such as an MKN-28 DNase or a HeLa DNase as described below.
The present invention relates to a DNase (hereinafter referred to as “MKN-28 DNase”) having the following properties:
(a) activity and substrate specificity: exhibiting an endonuclease activity;
(b) molecular weight: 48 to 43 kDa (determined by a gel filtration chromatography);
(c) optimum pH: pH 3.0 to 4.5;
(d) thermostability: the endonuclease activity is not inactivated by heating at 100° C. for 10 minutes; and
(e) susceptibility to proteinase K treatment: the endonuclease activity is inactivated by a treatment with proteinase K at 37° C. for 15 minutes.
The present invention relates to a DNase (hereinafter referred to as “HeLa DNase”) having the following properties:
(a) activity and substrate specificity: exhibiting an endonuclease activity;
(b) molecular weight: 63 kDa (determined by a gel filtration chromatography);
(c) optimum pH: pH 3.0 to 4.5;
(d) thermostability: the endonuclease activity is inactivated by heating at 100° C. for 10 minutes; and
(e) susceptibility to proteinase K treatment: the endonuclease activity is not inactivated by a treatment with proteinase K at 37° C. for 15 minutes.
The antitumor agent of the present invention exhibits an activity of inhibiting a cell proliferation with respect to various cancer cell lines, but does not exhibit the inhibitory activity to normal cells. The antitumor agent of the present invention specifically acts on cancer, and thus, has a few adverse effects.
The novel DNase according to the present invention, or a restriction enzyme which may be used as an active ingredient of the antitumor agent according to the present invention, exhibits an activity of inhibiting a cell proliferation with respect to various cancer cell lines, but does not exhibit the inhibitory activity to normal cells. Therefore, the novel DNase of the present invention or such a restriction enzyme is useful as an active ingredient of the antitumor agent according to the present invention.
[1] Antitumor Agent of the Present Invention
The antitumor agent of the present invention contains one or more DNases as an active ingredient. The DNase is not particularly limited, so long as it exhibits an activity of inhibiting a cell proliferation with respect to tumor cells, but does not exhibit the inhibitory activity to normal cells. As the DNase which may be used as an active ingredient of the antitumor agent according to the present invention, there may be mentioned, for example, the MKN-28 DNase of the present invention, the HeLa DNase of the present invention, DNase II, DNase I, NUC18, DNase V, DNase VI, a Ca2+/Mg2+ endonuclease (for example, human Ca2+/Mg2+ endonuclease, bovine Ca2+/Mg2+ endonuclease, or rat Ca2+/Mg2+ endonuclease), rat Mg2+ endonuclease, rat neutral DNase, bovine nuclear endonuclease, CHO acidic endonuclease, rat DNase α, rat DNase β, rat DNase γ, or various restriction enzymes. The DNase which may be used in the present invention includes not only an enzyme having a DNase activity alone, but also an enzyme having an enzyme activity other than the DNase activity together with the DNase activity, such as topoisomerase II (i.e., gyrase) or an integrase (for example, λ integrase).
The DNase as used herein includes an endonuclease and an exonuclease, and an endonuclease is preferred. The antitumor agent may contain only one DNase, or a combination of two or more DNases (for example, a combination of two or more endonucleases, a combination of two or more exonucleases, or a combination of one or more endonucleases and one or more exonucleases).
Whether or not a DNase exhibits an activity of inhibiting a cell proliferation with respect to tumor cells, but does not exhibit the inhibitory activity to normal cells may be easily judged, for example, by a known method for determining an antitumor activity [for example, an MTT method (J. Virol. Methods, 20, 309-321, 1988; or Journal of Virological Methods, 20, 309, 1988)].
In the MTT method, whether or not a DNase to be judged exhibits an activity of inhibiting a cell proliferation with respect to tumor cells, but does not exhibit the inhibitory activity to normal cells may be determined, for example, by the procedures described in Example 4. More particularly, cells for evaluation, i.e., a cancer cell line (for example, an MKN-28 cell or a HeLa cell) and a normal cell (for example, an MRC-5 cell or an HEF cell) are prepared as cell suspensions. An appropriately diluted series (for example, 1/2, 1/4, 1/8, 1/16, and 1/32) of a DNase solution are poured into each well of a microplate. After each cell suspension is further added into each well, the cells are cultivated for a predetermined period (for example, 4 days). After the cultivation, an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma chemical Co.] solution is added into each well. After an incubation for a predetermined period (for example, at 37° C. for 4 hours), a culture supernatant is removed from each well, and then an MTT formazan elution liquid [acidified isopropanol containing 10% (V/V) Triton X-100] is added into each well. After the plate is shaken, OD values are measured at wavelengths of 540 nm and 690 nm by a microplate reader, and each IC50 value is calculated. The IC50 value is indicated as a dilution of a test liquid or a concentration (for example, μg/mL) of a test drug which can inhibit a proliferation of a control cell (without an antitumor agent) to 50%. When the dilution is high, or when the drug concentration is low, it can be judged that the drug (substance) tested has a high activity.
As the DNase used in the present invention, the MKN-28 DNase or the HeLa DNase of the present invention, or a restriction enzyme is preferred.
It is known that a point mutation of a protooncogene located in a normal cell causes a cancer of normal cells to occur. For example, a human oncogene, activated c-ras (H-ras, K-ras, or N-ras), is a gene activated by a point mutation at a specific codon of a proto-ras gene.
For example, human lung cancer cell line A549 is a cancer cell in which the nucleotide sequence 5′-GGT-3′ (Gly) of the 12th codon in c-Ki-ras2 is changed to 5′-AGT-3′ (Ser): that is, the first base G among three bases in the codon is changed to A. In the A549 cell, the 11th to 12th nucleotide sequence in c-Ki-ras2 is GCTGGT before the mutation, and is GCTAGT after the mutation. The nucleotide sequence “CTAG” in the sequence GCTAGT after the mutation is a recognition sequence “C:TAG” (“:” means a cleavage site) of a restriction enzyme XspI. When the antitumor agent of the present invention is used with respect to a tumor cancer caused by the same point mutation as that in the A549 cell, for example, restriction enzyme XspI or an isoschizomer thereof may be selected as the active ingredient thereof. The restriction enzyme XspI does not cleave the nucleotide sequence GCTGGT before the mutation, and thus does not act on a normal cell.
In the present invention, an appropriate restriction enzyme may be selected on the basis of a type of tumor to be treated, that is, a nucleotide sequence containing a point mutation, for example, as shown in Table 1. In Table 1, numbers in parentheses in “Protocodon before mutation” are codon numbers. In “codon after mutation”, bases shown in lower case letters mean mutated bases, and recognition sites are underlined.
The antitumor agent of the present invention may contain the DNase alone, or preferably as a complex of the DNase and a liposome.
The liposome which may be used in the present invention is, for example, a liposome prepared from phospholipids, glycolipids, or lipid molecules (such as cholesterol) and/or surfactants. A unilamellar liposome or a multilamellar liposome may be effectively used.
The phospholipids may include, for example, glycerophospholipids (for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, or cardiolipin), or sphingophospholipids (for example, sphingomyelin, ceramide phosphorylethanolamine, or ceramide phosphorylglycerol).
The glycolipids may include, for example, glyceroglycolipids (for example, digalactosyldiglyceride or seminolipid), or sphingoglycolipids (for example, galactosylceramide or lactosylceramide).
The surfactants may include, for example, dicetyl phosphate or stearylamine.
The formulation of the antitumor agent according to the present invention containing a complex of the DNase and the liposome is not particularly limited, so long as the DNase and the liposome are contained in the antitumor agent as a complex. For example, the antitumor agent may be a mixture of the DNase and the liposome, or a formulation prepared by embedding the DNase in the liposome or encapsulating the DNase with the liposome. The embedded formulation is preferred.
The embedded formulation may be prepared, for example, in accordance with a method for embedding the DNase in the liposome. More particularly, a lipid (such as phosphatidylcholine, dicetyl phosphate, or cholesterol) is dissolved in an appropriate solvent (such as chloroform), and aliquots are poured into appropriate bottles or tubes. After the solvent is removed by blowing nitrogen gas thereinto, a DNase solution is further added and treated by a vortex mixer or the like. The whole is incubated at a predetermined temperature (for example, at 37° C.) for a predetermined period (for example, for 30 minutes) to obtain a liposome containing the DNase.
When the antitumor agent of the present invention contains the complex of the DNase and the liposome (hereinafter referred to as DNase/liposome complex), a thermally-denatured immunoglobulin G (aggregated IgG) may be further contained. The thermally-denatured IgG may be prepared, for example, by dissolving 15 mg of human IgG (Human IgG Purified; Sigma Chemical Co.) in 1 mL of Ringer solution and heating the solution at 60° C. for 10 minutes [Biochemistry, 15, 452, 1976]. To the DNase/liposome complex, a hundredth amount of the thermally-denatured IgG is added, and the mixture may be used, for example, at a concentration of 150 μg/mL (as the final concentration of the IgG).
It is known that the thermally-denatured IgG binds to an Fc receptor. Therefore, it is considered that the thermally-denatured IgG located on the surface of the liposome binds to the Fc receptor located on the membrane of a tumor cell and, as a result, the binding causes a receptor-mediated endocytosis to induce a higher activity of inhibiting cell proliferation [Biochemistry, 15(2), 452-460, 1976].
In the antitumor agent of the present invention, the DNase (preferably the DNase/liposome complex) may be administered alone or, optionally together with a pharmaceutically or veterinarily acceptable ordinary carrier or diluent, to a subject (an animal, preferably a mammal, particularly a human) in need of treatment or prevention of a cancer in an amount effective therefor. Furthermore, the DNase (preferably the DNase/liposome complex) as the active ingredient in the present invention may be used in the manufacture of an antitumor agent.
The formulation of the antitumor agent of the present invention is not particularly limited to, but may be, for example, oral medicines, such as powders, fine particles, granules, tablets, capsules, suspensions, emulsions, syrups, extracts or pills, or parenteral medicines, such as injections, liquids for external use, ointments, suppositories, creams for topical application, or eye lotions.
The oral medicines may be prepared by an ordinary method using, for example, fillers, binders, disintegrating agents, surfactants, lubricants, flowability-enhancers, diluting agents, preservatives, coloring agents, perfumes, tasting agents, stabilizers, humectants, antiseptics, antioxidants, such as gelatin, sodium alginate, starch, corn starch, saccharose, lactose, glucose, mannitol, carboxylmethylcellulose, dextrin, polyvinyl pyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, silicic anhydride, or synthetic aluminum silicate.
The parenteral administration may be, for example, an injection such as a subcutaneous, intravenous, or intraarterial injection, or a per rectum administration. Of the parenteral formulations, an injection is preferably used.
When the injections are prepared, for example, water-soluble solvents, such as physiological saline or Ringer solution, water-insoluble solvents, such as plant oil or fatty acid ester, agents for rendering isotonic, such as glucose or sodium chloride, solubilizing agents, stabilizing agents, antiseptics, suspending agents, or emulsifying agents may be optionally used, in addition to the active ingredient.
The antitumor agent of the present invention may be administered in the form of a sustained release preparation using sustained release polymers. For example, the antitumor agent of the present invention may be incorporated to a pellet made of ethylenevinyl acetate polymers, and the pellet may be surgically implanted in a tissue to be treated.
The antitumor agent of the present invention may contain the DNase (or the DNase/liposome comlex) in an amount of, but is by no means limited to, 0.01 to 99% by weight, preferably 0.1 to 80% by weight.
A dose of the antitumor agent of the present invention is not particularly limited, but may be determined dependent upon the kind of disease, the age, sex, body weight, or symptoms of the subject, a method of administration, or the like. The antitumor agent of the present invention may be orally or parenterally administered.
The antitumor agent of the present invention may be administered as a medicament or in various forms, for example, eatable or drinkable products such as health foods (preferably functional foods) or feeds. The term “foods” as used herein includes drinks.
As cancers which may be treated or prevented with the antitumor agent of the present invention, there may be mentioned, for example, stomach cancer, colon cancer, liver cancer, kidney cancer, breast cancer, oral cancer, pancreatic cancer, esophagus cancer, bladder cancer, uterine cancer, lung cancer, or leukemia.
[2] DNase of the Present Invention
Of the DNases which may be used as the active ingredient of the antitumor agent according to the present invention, the MKN-28 DNase and the HeLa DNase of the present invention are novel DNases.
The MKN-28 DNase of the present invention may be prepared from, for example, a human stomach cancer cell line MKN-28 (RCB1000, Riken), and has the following properties:
(a) Activity and substrate specificity: exhibiting an endonuclease activity
(b) Molecular weight: 48 to 43 kDa (determined by a gel filtration chromatography)
(c) Optimum pH: pH 3.0 to 4.5
(d) Thermostability: The endonuclease activity is not inactivated by heating at 100° C. for 10 minutes.
(e) Susceptibility to proteinase K treatment: The endonuclease activity is inactivated by a treatment with proteinase K at 37° C. for 15 minutes.
(f) Requirement for divalent cations: Ca2+ or Mg2+ is not required for the endonuclease activity. A slight dependency on Mn2+ or Zn2+ (0.01 to 1.0 mmol/L for Mn2+ and 0.01 to 0.1 mmol/L for Zn2+) is observed. Ca2+, Mg2+, Mn2+, or Zn2+ inhibits the endonuclease activity at a high concentration (10 mmol/L).
(g) Sensitivity to DNase inhibitors: Globular actin (G-actin) does not inhibit the nuclease activity.
Aurintricarboxylic acid (ATA) inhibits the nuclease activity.
Citrate inhibits the nuclease activity.
Iodoacetate inhibits the nuclease activity.
Sulfate ion (SO42−) inhibits the nuclease activity.
Spermine slightly inhibits the nuclease activity.
Ca2+, Mg2+, Mn2+, or Zn2+ inhibits the nuclease activity at a high concentration (10 mmol/L).
β-butyrolactone does not inhibit the nuclease activity.
1,3-butadienediepoxide does not inhibit the nuclease activity.
The MKN-28 DNase of the present invention may be prepared from the human stomach cancer cell line MKN-28 in accordance with, for example, the procedures described in Example 1 and Example 6(9). More particularly, the MKN-28 DNase of the present invention may be obtained by a process comprising the steps of:
(1) adding magnesium sulfate and ATP to an MKN-28 cell homogenate, and centrifuging the mixture to obtain a supernatant;
(2) salting out the supernatant obtained in the step (1) with 70% of ammonium sulfate, and centrifuging the whole to obtain a supernatant; and
(3) fractionating from the supernatant obtained in the step (2) a fraction having a molecular weight of 48 to 43 kDa by a gel filtration chromatography.
In the above step (3), for example, Sephacryl S-300 HR may be used to obtain the fraction of interest on the basis of the DNase activity as an index, in accordance with the procedures described in Example 2. The obtained fraction may be further purified on the basis of the DNase activity, for example, by an ion-exchange chromatography in accordance with the procedures described in Example 3.
The MKN-28 DNase of the present invention may be used as the active ingredient of the antitumor agent according to the present invention in the form of the purified DNase or a crude DNase [for example, the supernatant obtained in the step (1) or (2), or the fraction obtained in the step (3)].
The HeLa DNase of the present invention may be prepared from, for example, human cervical cancer cell line HeLa [RCB0007, Riken or ATCC CCL-2, American Type Culture Collection (ATCC)], and has the following properties:
(a) Activity and substrate specificity: exhibiting an endonuclease activity
(b) Molecular weight: 63 kDa (determined by a gel filtration chromatography)
(c) Optimum pH: pH 3.0 to 4.5
(d) Thermostability: The endonuclease activity is inactivated by heating at 100° C. for 10 minutes.
(e) Susceptibility to proteinase K treatment: The endonuclease activity is not inactivated by a treatment with proteinase K at 37° C. for 15 minutes.
(f) Requirement for divalent cations: Ca2+, Mg2+, Mn2+, or Zn2+ is not required for the endonuclease activity. Ca2+ or Mg2+ inhibits the endonuclease activity at a high concentration (10 mmol/L).
(g) Sensitivity to DNase inhibitors: G-actin does not inhibit the nuclease activity.
Aurintricarboxylic acid inhibits the nuclease activity.
Citrate does not inhibit the nuclease activity.
Iodoacetate inhibits the nuclease activity.
Sulfate ion (SO42−) inhibits the nuclease activity.
Spermine does not slightly inhibit the nuclease activity.
Zn2+ does not inhibit the nuclease activity.
β-butyrolactone does not inhibit the nuclease activity.
1,3-butadienediepoxide does not inhibit the nuclease activity.
The HeLa DNase of the present invention may be prepared from the human cervical cancer cell line HeLa in accordance with, for example, the procedures described in Example 1 and Example 6(9). More particularly, the HeLa DNase of the present invention may be obtained by a process comprising the steps of:
(1) adding magnesium sulfate and ATP to an HeLa cell homogenate, and centrifuging the mixture to obtain a supernatant;
(2) salting out the supernatant obtained in the step (1) with 70% of ammonium sulfate, and centrifuging the whole to obtain a supernatant; and
(3) fractionating from the supernatant obtained in the step (2) a fraction having a molecular weight of 63 kDa by a gel filtration chromatography.
In the above step (3), for example, Sephacryl S-300 HR may be used to obtain the fraction of interest on the basis of the DNase activity as an index, in accordance with the procedures described in Example 2. The obtained fraction may be further purified on the basis of the DNase activity, for example, by an ion-exchange chromatography in accordance with the procedures described in Example 3.
The HeLa DNase of the present invention may be used as the active ingredient of the antitumor agent according to the present invention in the form of the purified DNase or a crude DNase [for example, the supernatant obtained in the step (1) or (2), or the fraction obtained in the step (3)].
The DNase of the present invention exhibits an activity of inhibiting a cell proliferation with respect to cancer cell lines (for example, the MKN-28 cell or the HeLa cell), but does not exhibit the inhibitory activity to normal cells (for example, human fetal lung fibroblast MRC-5 or human fetal fibroblast HEF). Therefore, the DNase of the present invention is useful as the active ingredient of the antitumor agent according to the present invention.
Various properties of the MKN-28 DNase and HeLa DNase according to the present invention and known DNase II and DNase I are shown in Tables 2 and 3.
a)Determined by a gel filtration chromatography, except for DNase I. DNase I includes four types of molecules A, B, C, and D.
b)rat liver
c)porcine spleen
d)porcine liver
e)rat spleen
[inhb: inhibit]
The present invention will now be further illustrated by, but is by no means limited to, the following Examples.
In this example, human stomach cancer cell line MKN-28 (RCB1000, Riken) and human cervical cancer cell line HeLa (RCB0007, Riken or ATCC CCL-2, ATCC) were used to prepare cell extracts, and the resulting cell extracts were further fractionated, in accordance with the following procedures. For comparison, the same procedures were repeated except that human fetal lung fibroblast MRC-5 (RCB0211, Riken or ATCC CCL171, ATCC) was used as a normal cell.
More particularly, each cell line was cultivated in a Dulbecco's modified Eagle's medium, and monolayered cells after 3-day cultivation were used in the following procedures. Cells were washed with phosphate buffered saline (PBS), and collected with a cell scraper. Collected cells were suspended in PBS and centrifuged at 1200 rpm for 5 minutes. The washing treatment was repeated three times. Collected cells were suspended in 5 mL of PBS to prepare cell suspensions containing 8×107 cells (MKN-28 cell) and 1×108 cells (HeLa cell). Each cell suspension was treated with a supersonicator (Type UH-50; manufactured by MST) on ice for 5 minutes (20 kHz, 50 W) to disrupt cells. To each cell homogenate, magnesium sulfate (MgSO4) and ATP were added at final concentrations of 2 mg/mL and 10 mg/mL, respectively, and allowed to stand at 37° C. overnight (for 22 hours).
Each mixture was centrifuged at 3000 rpm at 4° C. for 30 minutes. The resulting supernatant was salted out with 70% of ammonium sulfate [(NH4)2SO4] (4.72 g of ground ammonium sulfate per 10 mL of supernatant). After the salting out at 4° C. for an hour, the whole was centrifuged at 1500 rpm at 4° C. for 15 minutes to separate a supernatant from a pellet. The resulting supernatant and pellet dissolved in PBS were dialyzed in PBS at 4° C. for 18 hours while stirring. The PBS was changed four times during the dialysis. After the dialysis, each solution was poured into cryotubes and kept at −20° C. In this connection, the above procedures were carried out as aseptically as possible. When each sample kept at −20° C. was used in the following procedures, the sample was centrifuged at 10000 rpm at 4° C. for 30 minutes and the resulting supernatant was used.
Hereinafter, the solution after the dialysis of the PBS solution containing the supernatant separated by the centrifugataion after the salting out is simply referred to as “the centrifugal supernatant”, and the solution after the dialysis of the PBS solution containing the pellet separated by the centrifugataion after the salting out is simply referred to as “the centrifugal pellet”.
The centrifugal supernatant prepared from the MKN-28 cells in Example 1 was fractionated under the following conditions.
Gel: Sephacryl S-300 HR (fraction range of globular proteins=1×104 to 1.5×106; Amersham, 17-0599-01) was used. A total gel bed was 114.8 mL [=(0.75 cm)2×3.14×65 cm].
Column: A column of 1.5 cm (diameter)×75 cm (height) (Econo-Column; Bio-Rad) was used.
Buffer: PBS (pH 7.2).
Flow rate: 0.4 mL/min.
Fraction: 2 mL/tube.
From collected fractions, DNase activity (+) fractions (i.e., fractions having a DNase activity) were selected, and an activity of digesting λDNA (Titer) in the selected fractions was assayed.
More particularly, an aliquot (i.e., undiluted solution) of each fraction was used to select DNase activity (+) fractions, and an activity of digesting λDNA in each positive fraction was titrated. The DNase activity and the titer were determined by an electrophoretic assay utilizing an activity of digesting λDNA as an index. Particularly, the titer was determined by diluting each fraction to four levels and analyzing an electrophoretic pattern (i.e., a degree of λDNA digestion) of λDNA digestion products.
The result is shown in Table 4. The peak of the DNase activity ranged between fractions No. 39 to No. 42, particularly fraction No. 40. The peak of the activity of digesting λDNA was fraction No. 40, and the titer thereof was 80-fold. Fractions No. 39 to No. 42 (four fractions) were used for the following purification step by an ion-exchange chromatography.
In this example, the DNase activity (+) fraction obtained in Example 2 were further purified by an ion-exchange chromatography using an Econo-Pac High Q cartridge (Stronganion, 732-0094; BIO-RAD).
Because the DNase activity (+) fraction obtained in Example 2 (i.e., a mixture of fractions No. 39 to No. 42) contained PBS as a buffer, PBS was replaced with a buffer for High Q by dialysis, and the resulting liquid was filtered through a membrane filter (0.45 μm). After confirming that the filtrate exhibited the DNase activity, the filtrate was applied to the Econo-Pac High Q cartridge. Elution was carried out by a concentration gradient of NaCl, that is, 50 mmol/L-Tris-HCl (pH7.5) containing 0.02 to 0.3 mol/L NaCl was used as a buffer for elution. The flow rate was 1.5 mL/2 min./fraction.
The DNase activity in each fraction was determined by an electrophoretic assay utilizing an activity of digesting λDNA as an index. As samples for the assay, an aliquot (i.e., undiluted solution) of each fraction was used. After the activity of digesting λDNA in each fraction was determined, the titer of DNase activity (+) fractions was determined to detect a peak of the DNase activity.
The result is shown in Table 5. The peak of the DNase activity ranged from fraction No. 5 (NaCl concentration=0.08 mol/L) to fraction No. 6 (NaCl concentration=0.10 mol/L).
In this example, an activity of inhibiting a cell proliferation in the centrifugal supernatants and the centrifugal pellets derived from various cancer cell lines and normal cells prepared in Example 1 was evaluated by an MTT method.
As cells for evaluation, the MKN-28 cell and the HeLa cell were used as cancer cell lines, and the MRC-5 cell was used as a normal cell. After each cell line was cultured for 3 days, cells were treated with trypsin, and dispersed cells were washed with PBS by centrifugation. Washed cells were suspended in a Dulbecco's modified Eagle's MEM medium containing 8% fetal calf serum (hereinafter referred to as GM), and counted with a hemocytometer. Each cell suspension was adjusted with the GM to cell densities of 5000 cells/50 μL and 2500 cells/50 μL (cancer cell lines) or 10000 cells/50 μL and 5000 cells/50 μL (MRC-5 cell).
To each well in a 96-well flat-bottomed plate, diluted series (undiluted, 1/2, 1/4, 1/8, 1/16, and 1/32; 50 μL/well) of each centrifugal supernatant or centrifugal pellet prepared in Example 1 were added. Further, each cell suspension was added to each well, and the plate was incubated at 37° C. in a CO2 incubator. After 3 or 4 days from the beginning of the incubation, 20 μL/well of an MTT solution was added to each well, and the plate was incubated at 37° C. for 3 hours. The culture supernatant was removed from each well, and 100 μL of an MTT formazan elution liquid was added to each well. After the plate was shaken at room temperature for 5 minutes, OD values were measured at wavelengths of 540 nm and 690 nm by a microplate reader, and each IC50 value was calculated.
As a control, PBS was used instead of the diluted series of each centrifugal supernatant or centrifugal pellet. For comparison, 5-fold-diluted series (100 μg/mL, 20 μg/mL, 4 μg/mL, 0.8 μg/mL, 0.16 μg/mL, and 0.032 μg/mL) of 5-fluorouracil (5-FU) were used instead of the diluted series of each centrifugal supernatant or centrifugal pellet.
The IC50 values when the cancer cell line MKN-28 was used as the cell for evaluation and the centrifugal supernatant and the centrifugal pellet derived from the MKN-28 cell were used as the cell fraction are shown in Table 6. Furthermore, the results of the control (PBS) and the comparison (5-FU) are shown in Table 6. Each IC50 value shown in Table 6 (or Tables 7 to 10 described below) is an average of plural (two to four) values. Each IC50 value is shown as a dilution (i.e., relative value) of a test liquid [or a concentration of a test drug (μg/mL for 5-FU)] which can inhibit a proliferation of a control cell (without an antitumor agent) to 50%.
As apparent from Table 6, the centrifugal supernatant and the centrifugal pellet derived from the MKN-28 cell exhibited an activity of inhibiting a cell proliferation with respect to the MKN-28 cell.
The IC50 values when the cancer cell line HeLa was used as the cell for evaluation and the centrifugal supernatant and the centrifugal pellet derived from the HeLa cell were used as the cell fraction are shown in Table 7. As apparent from Table 7, the centrifugal supernatant and the centrifugal pellet derived from the HeLa cell exhibited an activity of inhibiting a cell proliferation with respect to the HeLa cell.
The IC50 values when the normal cell MRC-5 was used as the cell for evaluation and the centrifugal supernatant and the centrifugal pellet derived from the MRC-5 cell were used as the cell fraction are shown in Table 8. As apparent from Table 8, the centrifugal supernatant and the centrifugal pellet derived from the MRC-5 cell did not exhibit an activity of inhibiting a cell proliferation with respect to the MRC-5 cell.
The IC50 values when three types of cells were used as the cell for evaluation and three types of centrifugal supernatants derived from each cell were used as the cell fraction are shown in Table 9 (3-day cultivation) and Table 10 (4-day cultivation).
As apparent from Tables 9 and 10, each centrifugal supernatant exhibited an activity of inhibiting a cell proliferation with respect to the cancer cell lines MKN-28 and HeLa, but did not exhibit the activity with respect to the normal cell MRC-5. The sensitivity to the centrifugal supernatants was as follows:
MKN-28 cell>HeLa cell>MRC-5 cell.
As shown in Example 6 described below, the centrifugal supernatants derived from the MKN-28 cell and the HeLa cell digested DNA, and thus were nucleases.
In this example, after the centrifugal supernatants derived from the cancer cell lines MKN-28 and HeLa, prepared in Example 1, were heated or treated with an RNase or a DNase, an activity thereof for inhibiting a cell proliferation with respect to the cancer cell line MKN-28 was examined to clarify the properties of each centrifugal supernatant. The activity of inhibiting a cell proliferation was measured in accordance with the method described in Example 4. The result (IC50 values) is shown in Table 11. The symbol “−” in Table 11 means that the measurement was not carried out.
The RNase treatment (“RN” in Table 11) was carried out by treating 100 μL of each cell extract with 10 μg of RNase (R 5125, Type III A; SIGMA) at 37° C. for 1 hour.
The DNase treatment (“DN” in Table 11) was carried out by treating 100 μL of each cell extract with 134 units of DNase (Lot 18600k; Nippon Gene) at 37° C. for 1 hour.
As a control for the RNase treatment and the DNase treatment, each cell extract was incubated at 37° C. for 1 hour (“37° C.” in Table 11).
To check the cell-proliferation inhibitory activity of the RNase or the DNase, a treatment with the RNase and PBS (“RN-Cont” in Table 11) and a treatment with the DNase and PBS (“DN-Cont” in Table 11) were carried out.
As the heating treatment, each cell extract was heated at 56° C. for 30 minutes (“56° C.” in Table 11).
As a control, each cell extract was treated with PBS (“PBS” in Table 11).
As shown in Table 11, the cell-proliferation inhibitory activity of each centrifugal supernatant derived cancer cell lines was not inactivated with the RNase or DNase treatment or by the heating treatment at 56° C. for 30 minutes. Furthermore, the activity was not inactivated with an RNase H treatment (data not shown). From the results, it was found that a factor showing the cell-proliferation inhibitory activity in each centrifugal supernatant derived cancer cell lines was (1) not a nucleic acid, and (2) not inactivated with the heating treatment. In this connection, the results depended on a reaction buffer, as shown in Example 6 described below.
[NT: not treated]
(1) Nuclease Activity Against λDNA and Genomic DNA of MKN-28 Cell
In this example, an activity of digesting DNA in each centrifugal supernatant derived from the cancer cell lines MKN-28 and HeLa prepared in Example 1 was examined.
Because each centrifugal supernatant contained PBS, PBS was replaced with a TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH8.0). As DNAs to be digested, λDNA (48502 bp; Takara) and a genomic DNA from the MKN-28 cell were used. The genomic DNA was extracted from the MKN-28 cell with a commercially available kit (Genomic Prep.™ cells and Tissue DNA Isolation Kit; Amersham pharmacia biotech.).
As reaction buffers, an L (Low) buffer (without NaCl), an M (Medium) buffer (50 mmol/L NaCl), and an H (High) buffer (100 mmol/L NaCl) were used. The reaction buffers were prepared by adding the above-mentioned amount of NaCl to a basic composition [10 mmol/L Tris-HCl (pH7.5), 10 mmol/L MgCl2, and 1 mmol/L DTT (Dithiothreitol)].
Each reaction was carried out at 37° C. for 1 hour, and a degree of DNA digestion was observed by electrophoresis.
The centrifugal supernatant derived from the MKN-28 cell digested both DNAs (i.e., λDNA and the genomic DNA derived from the MKN-28 cell) in the L or M buffer, but did not digest both DNAs in the H buffer.
The centrifugal supernatant derived from the HeLa cell did not digest both DNAs (i.e., λDNA and the genomic DNA derived from the MKN-28 cell) in the L, M, or H buffer.
(2) Optimum Buffer
The procedure described in Example 6(1) was repeated except that an A buffer [50 mmol/L Mes buffer (pH 5.8), 1 mmol/L CaCl2, and 3 mmol/L MgCl2] and a B buffer [50 mmol/L Mops buffer (pH 7.0), 1 mmol/L CaCl2, and 3 mmol/L MgCl2] were used as the reaction buffer.
The centrifugal supernatant derived from the HeLa cell digested both DNAs (i.e., λDNA and the genomic DNA derived from the MKN-28 cell) in the A buffer, but did not digest both DNAs in the B buffer.
The nuclease activity in the centrifugal supernatant derived from the MKN-28 cell was remarkable in the A buffer, and slightly digested λDNA in the B buffer.
(3) Resistance to Heating Treatment
Each centrifugal supernatant was heated at 100° C. for 10 minutes, and the nuclease activity was examined.
After the heating treatment, the centrifugal supernatant derived from the MKN-28 cell digested both DNAs (i.e., λDNA and the genomic DNA derived from the MKN-28 cell) in the L or M buffer. That is, the centrifugal supernatant derived from the MKN-28 cell was not inactivated by the heating treatment.
The centrifugal supernatant derived from the HeLa cell digested λDNA in the A buffer before the heating treatment, but did not digest λDNA after the heating treatment. That is, the centrifugal supernatant derived from the HeLa cell was inactivated by the heating treatment.
(4) Resistance to Proteinase K Treatment
Each centrifugal supernatant was treated with proteinase K at 37° C. for 15 minutes, and the nuclease activity was examined.
The centrifugal supernatant derived from the MKN-28 cell digested λDNA in the M buffer before the treatment with proteinase K, but did not digest λDNA after the treatment with proteinase K. That is, the centrifugal supernatant derived from the MKN-28 cell was inactivated by the treatment with proteinase K.
The centrifugal supernatant derived from the HeLa cell digested λDNA in the A buffer after the treatment with proteinase K. That is, the centrifugal supernatant derived from the HeLa cell was not inactivated by the treatment with proteinase K.
The results described in Example 6(1) to 6(4) are shown in Table 12.
[D: Digested, NI: Not inactivated, IA: Inactivated]
(5) Optimum pH
The optimum pH of the nuclease activity in each centrifugal supernatant was examined on the basis of the activity of digesting λDNA as an index.
λDNA was digested in 50 mmol/L buffers [Acetate-HCl (pH 1.0-5.5), MOPS-NaOH (pH 6.0-7.0), and Tris-HCl (pH 7.5-8.5); in increment of pH 0.5 from pH 1.0 to pH 8.5] supplemented with 1 mmol/L DTT at 37° C. for 30 minutes, and the digestion was observed by electrophoresis.
Both centrifugal supernatants (i.e., the centrifugal supernatant derived from the MKN-28 cell and the centrifugal supernatant derived from the HeLa cell) almost completely digested λDNA at pH 4.5 or less. When the same procedure was repeated except that no centrifugal supernatants were added, λDNA was digested at pH 2.5 or less. From the results, the optimum pH of each centrifugal supernatant was pH 3.0 to 4.5.
(6) Requirement for Divalent Cations
The requirement for divalent cations (Ca2+, Mg2+, Mn2+, and Zn2+) in each centrifugal supernatant was examined on the basis of the activity of digesting λDNA as an index.
After each centrifugal supernatant was dialyzed in PBS (Ca2+ and Mg2+ free), the titer thereof was measured to determine the optimum dilution concentration. As a result, the optimum dilution concentration of the centrifugal supernatant derived from the MKN-28 cell and the centrifugal supernatant derived from the HeLa cell were 1/5 and 1/3, respectively.
An acetate-HCl buffer (Ca2+ and Mg2+ free) supplemented with 3 mmol/L MgCl2 was used for examining the requirement for Ca2+. An acetate-HCl buffer (Ca2+ and Mg2+ free) supplemented with 3 mmol/L CaCl2 was used for examining the requirement for Mg2+. An acetate-HCl buffer (Ca2+ and Mg2+ free) supplemented was used for examining the requirement for Mn2+ or Zn2+. The pH of each buffer for the centrifugal supernatant derived from the MKN-28 cell was pH 4.5. The pH of each buffer for the centrifugal supernatant derived from the HeLa cell was pH 4.0.
The result in the centrifugal supernatant derived from the MKN-28 cell is shown in Table 13. The result in the centrifugal supernatant derived from the HeLa cell is shown in Table 14. In Table 13, the number of “+” indicates a degree of λDNA digestion, and “(−)” means that the assay was not performed. In addition, the results in the absence of Ca2+ and Mg2+ and the results in the absence of centrifugal supernatants (i.e., only buffer) were “+++” and “−” (not digested), respectively (data not shown).
As shown in Table 13, the centrifugal supernatant (1/5 dilution) derived from the MKN-28 cell required no divalent cations (Ca2+, Mg2+, Mn2+, or Zn2+), and Ca2+, Mg2+, or Zn2+ inhibited the activity at a high concentration (10 mmol/L or more).
In addition, the same procedure was repeated, except that the centrifugal supernatant derived from the MKN-28 cell was diluted to 1/40 (i.e., limited concentration for detecting the λDNA digestion), to confirm the requirement for divalent cations at a high sensitivity. Ca2+ or Mg2+ was not required. A slight dependency on Mn2+ (0.01 to 1.0 mmol/L) or Zn2+ (0.01 to 0.1 mmol/L) was observed. Ca2+, Mg2+, Mn2+, or Zn2+ inhibited the activity at a high concentration (10 mmol/L or more).
As shown in Table 14, the centrifugal supernatant (1/3 dilution) derived from the HeLa cell required no divalent cations (Ca2+, Mg2+, Mn2+, or Zn2+), and Ca2+ or Mg2+ inhibited the activity at a high concentration (10 mmol/L or more).
When the same procedure was repeated, except that the centrifugal supernatant derived from the HeLa cell was diluted to 1/20 (i.e., limited concentration for detecting the λDNA digestion), Ca2+, Mg2+, Mn2+, or Zn2+ was not required, and Ca2+, Mg2+, Mn2+, or Zn2+ inhibited the activity at a high concentration (10 mmol/L or more).
(7) Sensitivity to DNase Inhibitors
The sensitivity of each centrifugal supernatant to various inhibitors was examined on the basis of the activity of digesting λDNA as an index.
After the centrifugal supernatant derived from the MKN-28 cell was diluted to 1/5 to prepare a sample, the sample was incubated in the presence of each inhibitor at pH 4.5 at 37° C. for 30 minutes, and the λDNA digestion was tested by electrophoresis. The centrifugal supernatant derived from the HeLa cell was diluted to 1/3 to prepare a sample, the sample was incubated in the presence of each inhibitor at pH 4.0 at 37° C. for 30 minutes, and the λDNA digestion was tested by electrophoresis.
Effects of each inhibitor for λDNA digestion are shown in Table 15 and Table 16.
In Table 15, “(G)” means G-actin (Globular actin, derived from bovine muscle; Sigma), and the numbers “1,10,100” shown under “(G)” mean concentrations of G-actin (μg/mL). “(A)” means ATA (Aurintricarboxylic acid; Wako), and the numbers “1,10,100” shown under “(A)” mean concentrations of ATA (μmol/L). “(C)” means citrate (sodium citrate; Wako), “(I)” means Iodoacetate (Nakarai), “(SO)” means SO42− (MgSO4; Wako), “(Zn)” means Zn2+ (ZnCl2; Wako), and “(S)” means spermine. “(B)” means β-butyrolactone (Tokyo Kasei Kogyo), and the numbers “0.1,1.0,10” shown under “(B)” mean concentrations of β-butyrolactone (mmol/L). “(BD)” means 1,3-butadienediepoxide (Tokyo Kasei Kogyo), and the numbers “0.1,1.0,10” shown under “(B)” mean concentrations of 1,3-butadienediepoxide (mmol/L).
The number of “+” indicates a degree of the activity for inhibiting the λDNA digestion. “−” means no inhibitory activity of the λDNA digestion, and “±” means a slight inhibitory activity of the λDNA digestion.
(8) Digestion of Circular Double-Stranded DNA
To determine which the nuclease activity of each centrifugal supernatant was an endonuclease activity or an exonuclease activity, a circular double-stranded DNA (plasmid pACYC184; Nippon Gene, 13-0220) was treated with each centrifugal supernatant to examine the digestion as an index.
The centrifugal supernatant derived from the MKN-28 cell or the HeLa cell digested the plasmid pACYC184. The results showed that both centrifugal supernatants were endonucleases.
(9) Molecular Weight (Determined by a Gel Filtration Chromatography)
Each centrifugal supernatant was fractionated by a gel filtration chromatography to purify each DNase of the present invention and determine the molecular weight thereof. The conditions were as follows:
Column: A column of 1.5 cm (diameter)×75 cm (height) (Econo-Column; Bio-Rad) was used.
Gel: Sephacryl S-300 HR (fraction range of globular proteins=1×104 to 1.5×106; Amersham) was used. A total gel bed was 114.8 mL [=(0.75 cm)2×3.14×65 cm].
Buffer: PBS (pH 7.2).
Flow rate: 0.4 mL/min.
Fraction: 2 mL/tube.
Calculation of molecular weight: A commercially available kit (Gel Filtration Calibration Kits; Amersham Bioscience) was used. As molecular makers, albumin (M.W.=67000), ovalbumin (M.W.=43000), and chymotrypsinogen (M.W.=25000) were used.
After the fractionation, the titer of each fraction was measured on the basis of the activity of digesting λDNA as an index to determine the peak fraction of each DNase of the present invention.
When the centrifugal supernatant derived from the MKN-28 cell was used, the peak fraction was fractions No. 25 and No. 26 and the molecular weight thereof was 48 to 43 kDa.
When the centrifugal supernatant derived from the HeLa cell was used, the peak fraction was fraction No. 25 and the molecular weight thereof was 63 kDa.
The centrifugal supernatant derived from the MKN-28 cell was a DNase, as shown in Example 6. The optimum pH of the centrifugal supernatant derived from the MKN-28 cell (MKN-28 DNase) was acidic (pH 3.0 to 4.5), as previously determined by electrophoresis on the basis of the λDNA digestion as an index. Various acidic buffers having a pH acceptable to cell proliferation were used to prepare liposome formulations containing the MKN-28 DNase, and the activity thereof for inhibiting cell proliferation was examined.
The liposome was prepared by dissolving phosphatidylcholine (L-α-phosphatidylcholine, derived from egg yolk; Nakarai), dicetyl phosphate (Sigma), and cholesterol (ICN Biochemical Inc.) [7:2:1 (mole ratio)] in chloroform and evaporating chloroform.
More particularly, 70 mmol of phosphatidylcholine, 20 mmol of dicetyl phosphate, and 10 μmol of cholesterol was dissolved in 1 mL of chloroform (7:2:1; 100 μmol/mL). After the mixture was diluted with chloroform to 1/16 (6.25 μmol/mL), 50 μL of aliquots were added to sample bottles (0.3125 μmol/bottle). Nitrogen gas was blown into each sample bottle while rotating, to evaporate the chloroform, which was further evaporated under a reduced pressure to prepare a thin layer of liposome. To each sample bottle, 500 μL of a reaction buffer containing the DNase (or only a buffer) was added (0.3125 mmol/500 μL=0.625 μmol/mL).
As the reaction buffer contained together with the DNase in the liposome, various PBSs were used in view of isotonicity and composition. The PBS (pH 6.0) and the PBS (pH 7.2) may be easily prepared by changing a ratio of KH2PO4 and Na2HPO4 contained in PBS. Whether or not PBSs are appropriate for the reaction buffer was examined. Furthermore, whether or not the difference in pH of PBSs affects the activity of inhibiting cell proliferation was examined.
A membrane filter for concentration (Amicon) was used to concentrate the centrifugal supernatant derived from the MKN-28 cell, and the buffer was replaced with the PBS (pH 6.0) or the PBS (pH 7.2). To each sample bottle containing the thin layer of liposome, 500 μL of the PBS (pH 6.0 or 7.2) or the MKN-28-DNase-containing PBS (pH 6.0 or 7.2) was added and vortexed. A thermally-denatured human IgG (final concentration=150 μg/mL) was further added, and the whole was incubated at 37° C. for 30 minutes to prepare various thermally-denatured IgG coated (hereinafter sometimes referred to as “Agg-IgG coated”) liposome formulations containing the MKN-28 DNase (pH 6.0 or 7.2). The thermally-denatured human IgG was prepared by dissolving 15 mg of human IgG (Human IgG Purified; Sigma Chemical Co.) in 1 mL of Ringer solution and heating at 60° C. for 10 minutes.
The MKN-28 cell and a normal cell [human fetal fibroblast HEF (J. Infect. Dis., 163, 270-275, 1991)] were used to evaluate the cell-proliferation inhibitory activity of each Agg-IgG coated liposome formulation containing the MKN-28 DNase (pH 6.0 or 7.2) by the MTT method.
To each well in a 96-well microplate, double-diluted series (undiluted, 1/2, 1/4, 1/8, 1/16, and 1/32; 50 μL/well) of each Agg-IgG coated liposome formulation containing the MKN-28 DNase prepared with the GM were added. Further, 50 μL of a suspension of the MKN-28 cell or the HEF cell (5000 cells/50 μL) was added, and incubated at 37° C. in a CO2 incubator without changing the culture medium. After 4 days from the beginning of the incubation, each IC50 value was calculated by the MTT method.
As a control, double-diluted series of Agg-IgG coated liposome (without the MKN-28 DNase), double-diluted series of MKN-28 DNase (without the Agg-IgG coated liposome), and buffers [PBS (pH 6.0) and PBS (pH 7.2)] were used, instead of the double-diluted series of Agg-IgG coated liposome formulation containing the MKN-28 DNase.
The results are shown in Table 17 and Table 18. In Tables 17 and 18, “LP-DN”, “LP(-DN)”, “DN(-LP)”, and “PBS” mean the Agg-IgG coated liposome formulation containing the MKN-28 DNase, the Agg-IgG coated liposome (without the MKN-28 DNase), the MKN-28 DNase (without the Agg-IgG coated liposome), and only PBS (buffer), respectively. Each IC50 value shown in Tables 17 and 18 is an average of plural (two to four) values. The activities of inhibiting a proliferation of the cancer cell line MKN-28 and human fetal fibroblast HEF as a normal cell are shown in Table 17 and Table 18, respectively.
As to the IC50 values for the MKN-28 cell [when the PBS (pH 6.0) was used], the Agg-IgG coated liposome formulation containing the MKN-28 DNase [i.e., “LP-DN”] was 24.5, the Agg-IgG coated liposome (without the MKN-28 DNase) [i.e., “LP(-DN)”] was 4.3, the MKN-28 DNase (without the Agg-IgG coated liposome) [i.e., “DN(-LP)”] was 15.0, and only the PBS buffer [i.e., “PBS”] was 2.7.
As to the IC50 values for the MKN-28 cell [when the PBS (pH 7.2) was used], the Agg-IgG coated liposome formulation containing the MKN-28 DNase was 26.0, the Agg-IgG coated liposome (without the MKN-28 DNase) was 3.5, the MKN-28 DNase (without the Agg-IgG coated liposome) was 15.0, and only the PBS buffer was 2.5.
As to the IC50 values for the HEF cell [when the PBS (pH 6.0) was used], the Agg-IgG coated liposome formulation containing the MKN-28 DNase was 4.0, the Agg-IgG coated liposome (without the MKN-28 DNase) was 3.5, the MKN-28 DNase (without the Agg-IgG coated liposome) was <2.0, and only the PBS buffer was <2.0.
As to the IC50 values for the HEF cell [when the PBS (pH 7.2) was used], the Agg-IgG coated liposome formulation containing the MKN-28 DNase was 3.8, the Agg-IgG coated liposome (without the MKN-28 DNase) was 3.2, the MKN-28 DNase (without the Agg-IgG coated liposome) was <2.0, and only the PBS buffer was <2.0.
When the PBSs were used as the reaction buffer, the IC50 values for the MKN-28 cell were 2.7 (pH 6.0) and 2.5 (pH 7.2), and the IC50 values for the HEF cell were <2.0 (pH 6.0) and <2.0 (pH 7.2). The results show that PBSs have no cytotoxicity and are excellent for the reaction buffer, and that the difference between pH 6.0 and 7.2 does not affect the IC50 values.
When the liposome (without the DNase) was used, the IC50 values for the MKN-28 cell were 4.3 (pH 6.0) and 3.5 (pH 7.2), and the IC50 values for the HEF cell were 3.5 (pH 6.0) and 3.2 (pH 7.2). The concentration of liposome was 0.5 μmol/L or less. The results show that the liposome has no cytotoxicity at a concentration of not more than 0.5 μmol/L.
When the MKN-28 DNase (without the liposome) was used, the IC50 values for the MKN-28 cell were 15.0 (pH 6.0) and 15.0 (pH 7.2), and the IC50 values for the HEF cell were <2.0 (pH 6.0) and <2.0 (pH 7.2). The DNase alone exhibited the activity for inhibiting the proliferation of the MKN-28 cell, but did not exhibit the activity for inhibiting the proliferation of the HEF cell as a normal cell.
When the liposome-DNase was used, the IC50 values for the MKN-28 cell were 24.5 (pH 6.0) and 26.0 (pH 7.2), and the IC50 values for the HEF cell were 4.0 (pH 6.0) and 3.8 (pH 7.2). The liposome-DNase exhibited a high IC50 value for the MKN-28 cell, but had little cytotoxicity for the HEF cell as a normal cell.
The IC50 value of the liposome-DNase was superior to that of the DNase (without the liposome). The result shows that the increased activity can be obtained by embedding the DNase in the liposome.
As described above, the centrifugal supernatant as an active ingredient in the present invention may be applied to the use of an antitumor agent. Furthermore, a mixture thereof with the liposome is more effective as the active ingredient of the antitumor agent.
When a DNase acts on the DNA of a cancer cell, the DNase must be brought into direct contact with the DNA. As to a DNase located in cytoplasm, a nuclear membrane inhibits the contact. The nuclear membrane disappears only during the mitotic period (M phase). To examine the increased activity of the DNase for inhibiting a cell proliferation, various cultured cells in the M phase were prepared, and the inhibitory activity of the DNase-containing liposome with respect to the M-phase cells was examined.
A synthetic colchicine, colcemid (J. Radiat. Res., 14, 258-270, 1971) was used to prepare the M-phase cell. The optimum conditions for obtaining the living M-phase cell, and the conditions for evaluation the activity of inhibiting a cell proliferation by the MTT method were examined.
More particularly, cells were cultivated in a 25-cm2 culture flask (Falcon, 3014, 50 mL) for 3 days. After the mono-layered cells were washed, a growth medium supplemented with 0.025 μg/mL of colcemid (Nakarai, 09356-74) was added, and incubated at 37° C. for 6 hours. After the cells were washed gently, the culture flask was gently shaken to collect cells removed from the bottom of the flask. The collected cells were washed with the medium (GM) by centrifugation to remove colcemid. The washed cells were suspended in the growth medium and incubated.
The incubated cells were observed under a microscope at intervals of an hour; an appearance of mitotic cells is shown in Table 19. The appearance of mitotic cells was judged by an appearance of round cells as an index. Although almost cultured cells adhere to the glass surface and are thinly spread, mitotic cells become round and tend to leave the glass surface. Therefore, an appearance of round cells which tend to leave the monolayer was used as the index.
In Table 19, “−” means that the round cells appeared at a percentage of 0% with respect to the whole cells, “±” means that the round cells appeared at a percentage less than 5%, and “+” means that the round cells appeared at a percentage from 5% to less than 30%. The numbers in parentheses of “Round cells/flask” are numbers of positive cells strained by trypan blue.
The yields of colcemid-treated cells (number of cells after the treatment/number of cells before the treatment) were 1.4% (93,000/6,300,000) for the MKN-28 cell and 29% (153,000/523,000) for the MRC-5 cell.
Next, the cells obtained by the treatment with 0.025 μg/mL of colcemid at 37° C. for 6 hours were dispensed into each well in a 96-well plate to examine a growth activity of each mitotic cell. More particularly, when the mitotic MKN-28 cells (9300, 4650, 2325, and 1162 cells/well) were dispensed into each well and incubated at 37° C. for 5 days, it was found that the cell density of 9300 cells/well or more was preferable to the MTT assay. In this connection, the growth activity was slightly lowered. When the mitotic MRC-5 cells (15300, 7650, 3825, and 1912 cells/well) were dispensed into each cell, no monolayer specific for fibroblasts was formed (cells grew, but confusion was observed), but it is considered that the cell density of 7650 or 3825 cells/well may be used in the MTT assay.
From the above results, although the conventional MTT assay was generally carried out under the conditions in which cells were used at the cell density of 5000 cells/well and the cultivation was carried out at 37° C. for 4 days, it is considered that the MTT assay of the mitotic cells obtained by the colcemid treatment may be preferably carried out at the cell density of 10000 cells/well (MKN-28 cell) or 8000 cells/well (MRC-5 cells). In the MKN-28 cell, the growth activity was lowered in comparison with the normal cultured cell (i.e., not treated with colcemid), and dead cells were observed after 3 to 4 days from the beginning of the cultivation. In the MRC-5 cell, not only was the growth activity lowered, but also the activity of a cell proliferation was confused, and did not form the typical monolayer. The following MTT assay was carried out under the above conditions.
In this example, the purified DNase derived from the MKN-28 cell [that is, the purified DNase obtained by purifying the centrifugal supernatant obtained in Example 1 through the Sephacryl S-300 HR in accordance with the procedure described in Example 2, and further purifying the resulting fraction by the ion-exchange chromatography in accordance with the procedure described in Example 3] was used for preparing a liposome formulation, and the activity thereof for inhibiting the proliferation of the MKN-28 cell was examined.
More particularly, as the purified DNase derived from the MKN-28 cell, the peak fractions obtained in Example 3 having the DNase activity, i.e., fraction No. 5 [50 mmol/L Tris-HCl (pH 7.5)+0.08 mol/L NaCl] and fraction No. 6 [50 mmol/L Tris-HCl (pH 7.5)+0.10 mol/L NaCl] were used. The liposome was prepared in accordance with the procedure described in Example 7, except that the purified DNase obtained in Example 3 was used instead of the centrifugal supernatant obtained in Example 1, and that 50 mmol/L Tris-HCl (pH 7.5) supplemented with 0.08 mol/L NaCl was used as the reaction buffer, instead of the PBS. The MTT method was carried out in accordance with the procedure described in Example 7.
An amount of the purified MKN-28 DNase contained in the resulting Agg-IgG coated liposome formulation (suspension) containing the purified MKN-28 DNase was 17 units/100 μL. The “1 unit” as used herein means an amount of DNase capable of completely digesting 1 μg of λDNA at 37° C. for an hour. The amount of DNase contained in the liposome formulation was determined by centrifuging 100 μL of the liposome formulation to thereby remove the supernatant, adding 100 μL of the PBS solution containing 0.2% Triton X-100 to thereby dissolve the liposome, and evaluating the activity of the λDNA digestion. It was previously confirmed that Triton X-100 did not affect the DNase activity.
In this example, the activity of the purified MKN-28 DNase for inhibiting the proliferation of the MKN-28 cell or the MRC-5 cell was evaluated by a cell-suspension method and a monolayer method.
In the cell-suspension method, double-diluted series (undiluted, 1/2, 1/4, 1/8, 1/16, and 1/32; 50 μL/well) of the Agg-IgG coated liposome formulation containing the purified MKN-28 DNase prepared with the GM were dispensed into each well. Further, 50 μL of a suspension of the MKN-28 cell or the MRC-5 cell (5000 cells/50 μL) was added, and incubated at 37° C. in a CO2 incubator. After 4 days from the beginning of the incubation, each IC50 value was calculated by the MTT method.
As a control, double-diluted series of Agg-IgG coated liposome (without the purified MKN-28 DNase), double-diluted series of purified MKN-28 DNase (without the Agg-IgG coated liposome), and the reaction buffer were used, instead of the double-diluted series of Agg-IgG coated liposome formulation containing the purified MKN-28 DNase.
In the monolayer method, aliquots of a suspension of the MKN-28 cell or the MRC-5 cell (5000 cells/100 μL) were dispensed into each well of a 96-well microplate (100 μL/well), and cultured at 37° C. for 20 hours. After the culture medium was removed from each well, double-diluted series (1/2, 1/4, 1/8, 1/16, and 1/32; 100 μL/well) of the Agg-IgG coated liposome formulation containing the purified MKN-28 DNase prepared with the GM were dispensed into each well, and cultured at 37° C. After 4 days from the beginning of the incubation, each IC50 value was calculated by the MTT method.
As a control, double-diluted series of Agg-IgG coated liposome (without the purified MKN-28 DNase), double-diluted series of purified MKN-28 DNase (without the Agg-IgG coated liposome), and the reaction buffer (0.5 mmol/L Tris-HCl (pH 7.5) supplemented with 0.8 mol/L NaCl) were used, instead of the double-diluted series of Agg-IgG coated liposome formulation containing the purified MKN-28 DNase.
The result is shown in Table 20.
As shown in Table 20, when the MKN-28 cell was evaluated by the cell-suspension method, the IC50 value of the Agg-IgG coated liposome formulation containing the purified MKN-28 DNase [i.e., “LP-DN”] was 5.3, that of the Agg-IgG coated liposome (without the purified MKN-28 DNase) [i.e., “LP(-DN)”] was 2.7, that of the purified MKN-28 DNase (without the Agg-IgG coated liposome) [i.e., “DN(-LP)”] was 2.7, and that of only the reaction buffer was 2.8. From the result, the Agg-IgG coated liposome formulation containing the purified MKN-28 DNase apparently exhibited the activity of inhibiting the proliferation of the MKN-28 cell. In contrast, the inhibitory activity was not observed in the monolayer method. Furthermore, when the MRC-5 cell as a normal cell was used, each IC50 value was <2.0 by the cell-suspension method and the monolayer method, and the inhibitory activity was not observed.
The concentration of liposome was 0.5 mmol/L or less. As previously mentioned, liposome has no cytotoxicity at a concentration of 0.5 mmol/L or less, and it is considered that the result is not involved in the liposome concentration.
To enhance the activity of inhibiting a cell proliferation, cells to be treated were adjusted to the mitotic period in which the nuclear membrane disappeared (i.e., M phase), so that the DNase might be brought into contact with the DNA to be more easily digested. That is, cells were treated with a synthetic colchicine, colcemid, as described in Example 8.
More particularly, the MKN-28 cells or the MRC-5 cells were cultivated for 3 days, and each culture medium was changed to the growth medium supplemented with 0.025 μg/mL of colcemid. After the incubation at 37° C. for 6 hours, the culture flask was gently shaken to collect cells removed from the bottom of the flask. The collected cells were washed with the normal growth medium without colcemid. Double-diluted series (undiluted, 1/2, 1/4, 1/8, 1/16, and 1/32; 50 μL/well) of the Agg-IgG coated liposome formulation containing the purified MKN-28 DNase prepared with the GM were dispensed into each well of a 96-well microplate. Next, 50 μL of a suspension of the MKN-28 (M phase) cell (10000 cells/50 μL) or the MRC-5 (M phase) cell (8000 cells/50 μL) was added, and incubated at 37° C. in a CO2 incubator. After 4 days from the beginning of the incubation, each IC50 value was calculated by the MTT method.
As a control, double-diluted series of Agg-IgG coated liposome (without the purified MKN-28 DNase), double-diluted series of purified MKN-28 DNase (without the Agg-IgG coated liposome), and the reaction buffer were used, instead of the double-diluted series of Agg-IgG coated liposome formulation containing the purified MKN-28 DNase.
The result is shown in Table 21.
As shown in Table 21, when the MKN-28 cell treated with colcemid was used, the IC50 value of the Agg-IgG coated liposome formulation containing the purified MKN-28 DNase [LP-DN] was 9.3, and the activity of inhibiting the proliferation of the MKN-28 cell treated with colcemid was detected. In contrast, when the MRC-5 cell as a normal cell was used, each IC50 value was <2.0, and the inhibitory activity was not observed.
As described above, the DNase of the present invention may be applied to the use of an antitumor agent. Furthermore, a mixture thereof with the liposome is more effective as the active ingredient of the antitumor agent. Additionally, the use of the DNase (or the mixture thereof) together with colcemid is also effective.
In this example, a restriction enzyme XspI (Takara, 1095A) was used instead of the MKN-28 DNase to prepare a liposome formulation containing the restriction enzyme XspI, in accordance with the procedures described in Example 7, and the activity of inhibiting a cell proliferation was evaluated by the MTT method (the cell-suspension method or the monolayer method). As cells for the evaluation, human lung cancer cell line A549 (RCB0098, Riken or ATCC CCL185, ATCC), human stomach cancer cell line MKN-28, and human fetal lung fibroblast MRC-5 (as a normal cell) were used.
In protooncoges N-ras, Ha-ras, and Ki-ras, the codons at the 11th and 12th are GCT (Ala) and GGT (Gly), and the corresponding codons in the A549 cell are changed to GCT (Ala) and AGT (Ser). The nucleotide sequence “CTAG” in the sequence GCTAGT in the A549 cell is a recognition sequence “C:TAG” (“:” means a cleavage site) of the restriction enzyme XspI. A mutation which causes a transformation is not identified in the MKN-28 cell.
The results (IC50 values) are shown in Table 22 and Table 23. In Table 22, IC50 values are indicated as a dilution of liquid. In Table 23, IC50 values are indicated as a unit of “XspI units/well/100 μL”. In Tables 22 and 23, “LP-Xsp”, “LP(-Xsp)”, “Xsp(-LP)”, and “RB” mean the Agg-IgG coated liposome formulation containing the restriction enzyme XspI, the Agg-IgG coated liposome (without the restriction enzyme XspI), the restriction enzyme XspI (without the Agg-IgG coated liposome), and only the reaction buffer [20 mmol/L Tris-HCl (pH 8.5), 10 mmol/L MgCl2, 1 mmol/L DTT, and 100 mmol/L KCl], respectively.
As shown in Table 22, when the human lung cancer cell line A549 was evaluated by the cell-suspension method, the IC50 value of the Agg-IgG coated liposome formulation containing the restriction enzyme XspI was 12.0, and that of only the restriction enzyme XspI was 11.5. Both exhibited the activity of inhibiting the proliferation of the A549 cell.
When the human stomach cancer cell line MKN-28 was evaluated by the cell-suspension method, the IC50 value of the Agg-IgG coated liposome formulation containing the restriction enzyme XspI was 7.3, and that of only the restriction enzyme XspI was 7.2. When the same MKN-28 cell was evaluated by the monolayer method, the IC50 value of the Agg-IgG coated liposome formulation containing the restriction enzyme XspI was 15.0, and that of only the restriction enzyme XspI was 19.0. All cases exhibited the activity of inhibiting the proliferation of the MKN-28 cell.
In contrast, when the MRC-5 cell as a normal cell was used, the Agg-IgG coated liposome formulation containing the restriction enzyme XspI and the restriction enzyme XspI alone did not exhibit the activity of inhibiting the proliferation of the MRC-5 cell. From the result, it was confirmed that the antitumor agent of the present invention does not act on normal cells.
In this example, the activity of the Agg-IgG coated liposome formulation containing the restriction enzyme XspI for inhibiting a proliferation of each M-phase cell was evaluated in accordance with the methods described in Example 10. The results are shown in Table 24 and Table 25.
As shown in Tables 24 and 25, the antitumor agent of the present invention exhibited the activity of inhibiting a proliferation of human lung cancer cell line A549 or human stomach cancer cell line MKN-28, but did not act on normal cells.
As above, the present invention was explained with reference to particular embodiments, but modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-111349 | Apr 2005 | JP | national |
