WO2005115388A1

WO2005115388A1 - A therapeutic agent containing nicotinic acid or its derivatives as an effective ingredient for prevention and treatment of cancer

Info

Publication number: WO2005115388A1
Application number: PCT/KR2005/001399
Authority: WO
Inventors: Suk-Chul Bae; Won-Jae Kim
Original assignee: Biorunx Co., Ltd.
Priority date: 2004-05-12
Filing date: 2005-05-12
Publication date: 2005-12-08
Also published as: EP1753425A4; KR20060047820A; KR100775549B1; JP2007537246A; EP1753425A1; CN1953748A

Abstract

The present invention relates to a niacinamide or a derivatives therefrom, specifically, agents for preventing cancer and therapeutics comprising a niacinamide or a derivatives therefrom enhancing the expression of RUNX3 gene, cancer-suppressing gene which is inactivated by methylation. agents for preventing cancer and therapeutics of the present invention is used to cancer which relates to inactivation of RUNX3 such as gastric, lung, liver, larynx, colon, bladdar, prostate, pancreas, uterine and breast cancer.

Description

[DESCRIPTION] [invention Title] A THERAPEUTIC AGENT CONTAINING NICOTINIC ACID OR ITS DERIVATIVES AS AN EFFECTIVE INGREDIENT FOR PREVENTION AND TREATMENT OF CANCER

[Technical Field] The present invention relates to an agent for the prevention and the treatment of cancer containing nicotinic acid or its derivatives as an effective ingredient, more precisely, an agent 'for the prevention and the treatment of cancer containing nicotinic acid or its derivatives enhancing the expression of RUNX3 gene, a cancer suppressor gene that is inactivated by methylation, and activating the gene by inhibiting the decomposition of RUNX3 protein by preventing deacethylation of RUNX3 protein, as an effective ingredient . [Background Art! Three major treatment methods of cancer, today, are surgical operation, chemotherapy and radiotherapy. And in fact, ultimodality approach combining the above methods with laser therapy, etc, is being performed. Considering pain by the treatment or possibility of metastasis, chemotherapy is most expected, among them. Thus, in variety of anticancer agents have been developed and clinically used to aid chemotherapy. Those agents have been developed based on the principal of killing actively divided cells selectively. However, those agents kill not only cancer cells but also actively divided normal cells such as immune cells, hair follicle cells, etc, causing serious side effects, so that long-term administration of those agents is not available. Thus, it is important to develop a novel anticancer agent based on the fundamental treatment of a cause of cancer.

While the activation of oncogenes induces abnormal proliferation of cells, tumor suppressor genes inhibit the abnormal proliferation of cells and sometimes block the generation of tumor cells by operating cell death program destroying specific target cells. Even when an abnormally activated oncogene is included in a cell, the cell cannot be growing to a tumor cell by the normal action of a tumor suppressor gene. Thus, to generate a tumor cell, the activation of an oncogene and the inactivation of a tumor suppressor gene should occur in a cell simultaneously. The injudicious cell proliferation, one of the characteristics of a tumor cell, was confirmed to be resulted from simultaneous activation of an oncogene and inactivation of a tumor suppressor gene. Particularly,

TGF- β dependent apoptosis in a normal cell plays an important role in defense against tumor cells. At this time, inactivation of a tumor suppressor gene promotes cell division and inhibits TGF- β dependent apoptosis, resulting in the generation of a tumor cell. One of the key points causing inactivation of a tumor suppressor gene is abnormal DNA methylation in CpG island (Jones and Laird, Nature genet. 21, 163-167, 1999) . The methylation in CpG island is induced by DNA methylase. When such methylation goes on, MeCP2 protein binds to the methylated DNA, leading to the concentration of histone deacethylase . Histone deacethylase, then, removes acethyl group attached on histone, and as a result, DNA structures around are denser, leading to the inhibition of gene expression. The gene inhibited by DNA methylation can be expressed again by the activity of a DNA methylase inhibitor or a histone deacethylase inhibitor. Acethylation/deacethylation occurs not only in histone but also in some transcription factors such as p53, RUNX3, etc. For example, when acethylation in RUNX3 is induced, protein decomposition by ubiquitination is suppressed but the activity of RUNX3 is promoted (Jin et al . , J. Biol . Chem. 2004, Vol. 279, pp. 29409-17) . That is, the promotion of RUNX3 activity is caused by the inhibition of deacethylation. Thus, acethylation/deacethylation is a very important mechanism that regulates the function of a protein. As a matter of fact, it has been recently reported that the expression of RUNX3, which is a tumor suppressor gene confirmed previously by the present inventors, is inhibited by DNA methylation in various human cancers, for example, according to those reports, RUNX3 expression is 60% inhibited in stomach cancer (Li QL et al., .Cell. 2002 109 (1) : 113-24; Oshimo Y. et al . , Pathobiology. 2004, 71 (3) : 137-43) , 25-40% inhibited in lung cancer (LI QL et al . BBRC. 2004, 314 (1) : 223-8; Kim TY et al., Lab Invest. 2004, 84 ( 4 ) : 479-84 ) , 62% inhibited in laryngeal cancer, 25% inhibited in breast cancer (Kim TY et al.., Lab Invest. 2004, 84 (4 ) : 479-8 ) , 70% inhibited in pancreatic cancer (Li J et al., J Clin Pathol. 2004 57(3):294-9 ; Wada M et al . , Oncogene. 2004, 23(13): 2401-7), 73% inhibited in hepatocellular carcinoma (Xiao WH and Liu WW, World J Gastroenterol . 2004, 10 (3) :376-80; Kim TY et al . , Lab Invest. 2004, 84 (4) : 479-84 ) , 75% inhibited in cholangiocarcinoma (Wada M et al., Oncogene. 2004, 23(13): 2401-7), 23% inhibited in prostatic cancer (Kang GH et al . , J. Pathol. 2004, 202 (2) :233-40; Kim TY et al . , Lab Invest. 2004, 84 (4) : 479-84) , and 45% inhibited in colon carcinoma (Ku JL et al., Oncogene, 2004, 23, 6736-6742).

In addition, the activation of RUNX3 gene was proved to remarkably inhibit the tumor cell proliferation (Li et al., Cell, 109(1), 113-24, 2002; Balmain, Nature 417(6886), 235-7, 2002). Based on the provement, the development of an agent for the prevention and the treatment of cancer has been focused on the study of the activation of RUNX3 gene.

Thus, the present inventors have endeavored to find out a factor that is able to activate RUNX3, a tumor suppressor gene, inactivated in a cancer cell in vivo .

As a result, the present inventors found the fact that nicotinic acid and its derivatives could activate RUNX3 gene, which was inactivated by methylation, and increased the amount of RUNX3 protein. And further the present inventors completed this invention by confirming that the nicotinic acid and its derivatives can be effectively used for the prevention and the treatment of cancer. [Disclosure] [Technical Problem] It is an object of the present invention to provide an agent for the prevention and the treatment of cancer containing nicotinic acid or its derivatives as an effective ingredient, which is able to activate RUNX3 gene, a tumor suppressor gene inactivated by methylation in a tumor cell, and promote the activity of RUNX3 protein derived from the gene.

[Technical Solution] To achieve the above object, the present invention provides an agent for the prevention and the treatment of cancer containing nicotinic acid or its derivatives as an effective ingredient.

Hereinafter, the present invention is described in detail . The present invention provides an agent for the prevention and the treatment of cancer containing nicotinic acid or its derivatives as an effective ingredient . The present inventors expected for an agent that could re-activate the inactivated tumor suppressor gene RUNX3 in vivo to have an anticancer effect. Thus, the inventors have searched a substance that is able to activate RUNX3 gene, and as a result, have confirmed that niacinamide can activate RUNX3. The present inventors investigated whether or not RUNX3, a tumor suppressor gene, was methylated in tumor tissues to examine the relation of tumor progression and methylation in a cancer patient. As a result, RUNX3 gene was frequently methylated in tissues of cancer patients and those patients group showing methylation of RUNX3 was proved to have high rates of recurrence of cancer and metastasis (see Table 1 - Table 3). The above results indicate that it is one way to prevent the progression of cancer to reactivate RUNX3 inactivated by methylation. And the idea forced present inventors to search a substance enabling the activation of RUNX3 gene, as an effort of detecting an agent to hinder the development and the progression of cancer. As a result, it was confirmed that niacinamide can reactivate RUNX3 inactivated by methylation (see Fig. 2). It was also investigated whether or not niacinamide was able to increase the amount of RUNX3 protein. As a result, the amount of RUNX3 protein was reduced as RUNX3 was decomposed by deacetylation induced by histone deacethylase, however, the administration of niacinamide brought the increase of acetylation of RUNX3 protein, resulting in the increase of the amount of the protein (see Fig. 3 and Fig. 4) . In order to confirm the in vivo anticancer effect of niacinamide, the inhibition of tumor formation was observed in a mouse with cancer induced by a carcinogen. N-butyl-N-4- (hydroxybutyl) -Nitrosamine (BBN) was used as a carcinogen herein, and the inhibition of tumor formation was analyzed by comparing the tumorigenesis between the mice provided with BBN solution as a drinking water for 20 weeks and the mice provided with BBN and niacinamide solution as a drinking water for 20 weeks (see Fig. 5 and Fig. 6) . As a result, in BBN treating group, methylation is induced in CpG island of RUNX3 exon 1 region, meaning the activity of RUNX3 was inhibited. So, bladder cancer was developed 100% in that group, and 80% of them were infiltrating cancer and 23% were superficial cancer. On the contrary, in BBN and niacinamide treating group, methylation was remarkably reduced in CpG island of RUNX3 exon 1 region, resulting in the decrease of bladder cancer development

(58%), and malignicity of cancer was also reduced. So, only 23% of them were infiltrating cancer and 77% of them were superficial cancer. The above results indicate that niacinamide inhibits the inactivation of RUNX3 caused by BBN, suggesting that it can inhibit tumorigenesis by BBN. From the above results, it was confirmed that niacinamide of the present invention can reactivate RUNX3 inactivated by methylation and thus, inhibit tumor growth significantly. In accordance with the above results, the tumor suppressor gene RUNX3 was found, in fact, inactivated by methylation in variety of human cancers such as stomach cancer, lung cancer, laryngeal cancer, breast cancer, pancreatic cancer, hepatocellular carcinoma, cholangiocarcinoma, prostatic cancer, colon carcinoma, etc (Li QL et al., Cell. 2002 109 (1) : 113-24; Oshimo Y. et al., Pathobiology. 2004, 71 (3) : 137-43) ; LI QL et al . BBRC. 2004, 314 (1) :223-8; Kim TY et al . , Lab Invest. 2004, 84 (4) :479-84; Li J et al . , J Clin Pathol. 2004 57(3):294-9; Wada M et al . , Oncogene. 2004, 23(13): 2401-7; Xiao WH and Liu WW, World J Gastroenterol . 2004, 10 (3) :376-80; Kang GH et al . , J. Pathol. 2004, 202 (2) :233-40; Ku JL et al . , Oncogene, 2004, 23, 6736- 6742), and the activation of RUNX3 gene was proved to inhibit cancer cell proliferation remarkably (Kim et al., Lab Invest. 84(4), 479-84, 2004; Wada et al . , Oncogen, 23(13), 2401-7, 2004; Li et al . , Biochem Biophys Res Commun, 314(1). 223-8, 2004; Li et al . , Cell, 109(1), 113-24, 2002; Li et al . , Cell, 109(1), 113-24, 2002; Bal ain, Nature, 417(6886), 235-7, 2002). Therefore, the pharmaceutical composition containing nicotinic acid or its derivatives as an effective ingredient, of the present invention, can reactivate RUNX3 gene, so the composition can be effectively used for the prevention and the treatment of many cancers induced by inactivation of RUNX3, for example stomach cancer, lung cancer, hepatoma, laryngeal cancer, colorectal cancer, bladder cancer, prostatic cancer, pancreatic cancer, uterine cancer or breast cancer. Nicotinic acid or its derivatives can be used as an effective ingredient of an agent for the prevention and the treatment of cancer of the present invention, and the nicotinic acid derivatives can include niacinamide, nicotinic acid, 6-aminonicotinamide, isonicotinamide, isonicotinohydrazide, N-methylnicotinamide, nicorandil, pyridine-2-carboxamide, thionicotin amide or 6- chloronicotinamide . The pharmaceutical composition of the present invention containing nicotinic acid derivatives as an effective ingredient can be administered orally or parenterally and be used in general forms of pharmaceutical formulation. The pharmaceutical composition of the present invention can be prepared for oral or parenteral administration by mixing with generally used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients. Solid formulations for oral administration are tablets, pills, dusting powders, granules and capsules. These solid formulations are prepared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above-mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin. Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions, and suppositories. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc. Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc.

The effective dosage of nicotinic acid derivatives of the present invention is 1-50 mg/kg, and more preferably 10-30 mg/kg, and the administration frequency is 1-3 times a day.

Acute toxicity test with nicotinic acid derivatives was performed in mice. Any change was not observed upto the dosage of 2 g/kg in a group treated with nicotinic acid derivatives and any significant side effect was not observed, either, compared with a group administered with water only. The present invention also provides a prevention and/or treatment method for cancer including the step of administrating nicotinic acid derivatives to a target subject. The present invention further provides a usage of nicotinic acid derivatives for the preparation of an agent for the prevention and/or the treatment of cancer.

[Description of Drawings] Fig. 1A is an electrophoresis photograph showing that RUNX3 gene is not methylated in normal tissues, which is confirmed by PCR, M: Methylated DNA U: Unmethylated DNA

Fig. IB is an electrophoresis photograph showing that RUNX3 gene is methylated in bladder cancer tissues, which is confirmed by PCR, M: Methylated DNA U: Unmethylated DNA

Fig. 2 is an electrophoresis photograph showing the activation of methylated RUNX3 by the treatment of niacinamide,

Fig. 3 is a photograph showing that the amount of RUNX3 protein is reduced by the combination with histone deacetylase (Sirt2),

Fig. 4 is a photograph showing that the decrease of RUNX3 protein by histone deacetylase (Sirt2) is related with the inhibition of acetylation of RUNX3, and reversely, acethylation of intracellular RUNX3 protein and the resultant increase of RUNX3 protein are induced by the administration of niacinamide,

Fig. 5 is a photograph of bladder cancer tissues observed by naked eye showing the comparison of tumorigenesis between a group provided with N-butyl-N-4- (hydroxybutyl) -Nitrosamine (BBN) solution, a carcinogen, as a drinking water for 20 weeks and the other group provided with BBN and niacinamide solution as a drinking water for 20 weeks,

Fig. 6A is a photomicrograph showing bladder tissues of mice provided with BBN solution, a carcinogen, as a drinking water for 20 weeks, Fig. 6B is a photomicrograph showing bladder tissues of mice provided with BBN and niacinamide solution as a drinking water for 20 weeks.

[Best Mode] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

<Example 1> Correlation between RUNX3 methylation and tumorigenesis In order to confirm whether or not RUNX3 gene was methylated in cancer patient tissues, tissues of a bladder cancer patient were extracted and methylation therein was investigated. Particularly, genomic DNA was extracted from both normal bladder tissues and bladder cancer tissues, which was then purified, followed by the treatment with 3 M NaS0₂ (sodium bisulfite) at 50^°C for 16 hours. During the above process, cytosine was changed into uracil in DNA which was not methylated. After the treatment, the DNA was purified again. Then, PCR was performed using two primer sets prepared based on the sequence of CpG island of RUNX3 genomic DNA; one primer set consisting of SEQ. ID. No 1 and SEQ. ID. No 2 was for amplification of methylated DNA and the other primer set consisting of SEQ. ID. No 3 and SEQ. ID. No 4 was for amplification of non-methylated DNA. PCR was performed as follows; denaturation at 94 ^°C for 14 seconds, annealing at 60 °C for 50 seconds, polymerization at 72^°C for 58 seconds, 40 cycles from denaturation to polymerization. PCR using the above primers can confirm whether or not RUNX3 gene is inactivated by methylation. If DNA is amplified with the primer set of SEQ. ID. No 1 and SEQ. ID. No 2, it means RUNX3 is inactivated by methylation. Likewise, if DNA is amplified with the primer set of SEQ. ID. No 3 and SEQ. ID. No 4, it means RUNX3 is not methylated. As shown in Fig. 1, methylation was not observed in promoter region of RUNX3 gene in normal bladder tissues extracted from 20 normal individuals. In the meantime, 74% of methylation (88 out of 119 individuals) was observed in bladder cancer patient tissues. In respect of bladder cancer recurrent rate, while patients having RUNX3 gene not methylated in their cancer tissues showed 61.11% recurrent rate, those having RUNX3 gene methylated in their cancer tissues showed 88% recurrent rate. That is, the chance of cancer recurrence is very high in those patients having methylated RUNX3 gene (Table 1) . [Table l] Correlation between RUNX3 methylation and cancer recurrent rate

p=0.0036, OR=4.6667, 95% CI=1.5774-13.8064 As shown in Table 2, cancer progression was speedy in cancer tissues in which RUNX3 was methylated. 94.74% of the patients with advanced cancer had methylated RUNX3 gene, indicating that methylation of RUNX3 gene plays an important role in the progression of cancer.

[Table 2] Correlation between RUNX3 methylation and cancer progression

p=0.0155, OR=9.00, 95%CI=1.1236-72.0598

In conclusion, methylation of RUNX3 gene in cancer cells increased tumorigenesis 109 times and also increased cancer recurrent rate and cancer progression 5 times and 9 times, respectively, compared with cancer cells in which methylation of RUNX3 gene was not induced.

<Example 2> Activation of RUNX3 by niacinamide Based on the results obtained from the above <Example 1>, the present inventors searched a substance being able to inhibit the methylation of tumor suppressor gene RUNX3, and as a result, the inventors confirmed that niacinamide and its derivatives could inhibit the methylation of RUNX3 gene. Niacinamide was treated to the culture solution of cancer cell line in which RUNX3 gene was inactivated by DNA methylation. The treatment was continued for 4 days and the final concentration was adjusted to 1 mg/ml. Reverse transcriptase polymerase chain reaction (RT-PCT) was performed using a primer set of Ps-CA represented by SEQ. ID. No 5 and Ps-CB represented by SEQ. ID. No 6 to detect the expression of RUNX3 gene. As shown in Fig. 2, niacinamide was confirmed to reactivate RUNX gene, inactivated by methylation, in stomach cancer cell lines SUN16 and MKN74, lung cancer cell lines NC1-H226, NC1- H460 and Sq-1, and bladder cancer cell line T24.

<Example 3> Decomposition of RUNX3 protein by deacethylation In order to investigate the correlation between deacethylation and decomposition of RUNX3 protein, genes each expressing Sirt2 (type III Histone deacetylase) and RUNX3 were inserted into a cell line expressing Sir2 and RUNX3, independently or together. Immunoprecipitation and Western blotting were performed to investigate the effect of the insertion (Janeway et al . , Immunobiology . 5th ed. Garland Publishing, 2001) . As a result, Sirt2 protein was detected from the immunoprecipitation (IP: RUNX3, IB: Sirt2) with RUNX3, and RUNX3 protein was detected from the immunoprecipitation (IP: Sirt2, IB: RUNX3) with Sirt2 (Fig. 3), suggesting that RUNX3 and Sirt2 are physically linked. It was additionally confirmed that the amount of RUNX3 protein was reduced by Sirt2 (Fig. 3, IB: RUNX3) .

<Example 4> Increase of the amount of RUNX3 protein by niacinamide Intracellular expressions of RUNX3, acetyltransferase (p300) and deacetylase (Sirt2) were induced and niacinamide was added to the cell culture medium, to investigate the change of RUNX3 protein level by niacinamide. Immunoprecipitation and Western blotting were performed for the investigation. As a result, the diminution of RUNX3 protein (Fig. 4, Lane 1 and 2) caused by the expression of Sirt2 was recovered by the administration of niacinamide (Fig. 4 Lane 3, 4 and 5) .

<Example 5> In vivo anticancer effect of niacinamide In order to confirm the anticancer effect of niacinamide, tumorigenesis inhibition test was performed by using mice with cancer induced by a carcinogen. Mice were divided into two groups; one was provided with 0.05% (w/w) N-butyl-N-4- (hydroxybutyl) -Nitrosamine (BBN) solution, a carcinogen, as a drinking water for 20 weeks and the other group was provided with 0.05% BBN and 1% (w/w) niacinamide (10 mg/ l ) solution as a drinking water for 20 weeks. And the comparison was made between the two groups. C3H/HE female mice at 6 weeks were used as test animals herein. Control group mice were provided with water or 1% (W/W) niacinamide solution for 20 weeks. On the final day of experiment, the mice were sacrificed and bladders were extracted to investigate methylation of RUNX3 gene and the formation and the size of a tumor. The development of bladder cancer in experimental group was observed by the naked eye and microscope, as shown in Fig. 5 and Fig. 6A. Precisely, BBN induced methylation in CpG island of RUNX3 exon 1 region, resulting in the inhibition of the activity of RUN 3. Thus, bladder cancer was developed 100% in the group treated with BBN. In particular, 80% of cancer induced by BBN was infiltrating cancer, and 20% of them were superficial cancer (Table 3).

[Table 3] Carcinogenic profile of 0.05% BBN treating group

Methylation in CpG island of RUNX3 exon 1 region was remarkably reduced in the group treated with BBN and niacinamide together. In the group, development rate of bladder cancer was lowered to 58% (Fig. 5 and Fig. 6B) and malignancy was also decreased (23% of the cancer were infiltrating and 77% were superficial) (Table 4). The above results indicate that niacinamide inhibits inactivation of RUNX3 by BBN, meaning that niacinamide can inhibit tumorigenesis induced by BBN.

[Table 4] Carcinogenic profile of 0.05% BBN and niacinamide treating group

<Example 6> Inhibition of in vivo methylation by nicotinic acid and its derivatives The present inventors investigated the inhibition of in vivo methylation by nicotinic acid and its derivatives and the results are as follows.

[Table 5] Inhibition of in vi vo methylation by nicotinic acid derivatives

Methylation of RUNX3 gene was 100% induced in the group treated with BBN, but the methylation was inhibited, as shown in Table 4, in the group treated with BBN and nicotinic acid or its derivatives together. Therefore, the nicotinic acid or its derivatives are believed to have anticancer activity.

<Example 7> Acute toxicity test in mice via oral administration The following experiments were performed to see if the pharmaceutical composition of the present invention containing nicotinic acid or its derivatives as an effective ingredient has acute toxicity in mice (C3H/HE 6) . The pharmaceutical composition of the present invention was orally administered to 2 mice per group, as a drinking water, at the dosage of 2 g/kg everyday for 5 months. Death, clinical symptoms, and weight change in mice were observed, hematological tests and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen were checked with eyes during autopsy. The results showed that the pharmaceutical composition of the present invention did not cause any specific clinical symptoms, weight change, or death in rats. No change was observed in hematological tests, biochemical tests of blood, and autopsy. Therefore, the pharmaceutical composition used in this experiment was evaluated to be a safe substance since it did not cause any toxic change in mice up to the level of 2 g/kg and its estimated LD50 value is much greater than 5 g/kg in mice .

[industrial Applicability] The present invention relates to an agent for the prevention and the treatment of cancer containing nicotinic acid or its derivatives as an effective ingredient, more precisely, an agent for the prevention and the treatment of cancer containing nicotinic acid and its derivatives, which are able to reactivate RUNX3 gene inactivated by methylation, as an effective ingredient. The agent for the prevention and the treatment of cancer of the present invention can be effectively used for the treatment and the prevention of solid cancer related to the inactivation of RUNX3 gene such as stomach cancer, lung cancer, hepato a, laryngeal cancer, colorectal cancer, bladder cancer, prostatic cancer, pancreatic cancer, uterine cancer or breast cancer. Unlike the conventional anticancer agents designed to kill cells showing very active cell division, the agent of the present invention is developed based on the principle of activating a tumor suppressor gene in a cancer cell, making it as a promising candidate for a novel anticancer agent with less side effects.

[Sequence List Text! SEQ. ID. No 1 and SEQ. ID. No 2 are primers for amplification of methylated DNA, SEQ. ID. No 3 and SEQ. ID. No 4 are primers for amplification of non-methylated DNA, SEQ. ID. No 5 is a Ps-CA primer, SEQ. ID. No 6 is a Ps-CB primer. Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

[CLAIMS] [Claim l] An agent for the prevention and the treatment of cancer containing nicotinic acid or its derivatives as an effective ingredient having the following formula:

Wherein, Ri is oxygen or sulfur, R₂ is H, OH, Cι~Cι₀ alkyl, NH₂, NH-NH₂, NH-C₂H₄-0-N0₂- or OR₃, R₃ is Cι~C₅ alkyl, X is H, NH₂ or halogen atom, and Y is H or CH₃.

[Claim 2] The agent for the prevention and the treatment of cancer as set forth in claim 1, wherein the Ri is Ci-Cs alkyl .

[Claim 3] The agent for the prevention and the treatment of cancer as set forth in claim 1, wherein the halogen atom is F or CI.

[Claim 4] The agent for the prevention and the treatment of cancer as set forth in claim 1, wherein the R₂ is CH₃ or C₂H5.

[Claim 5] The agent for the prevention and the treatment of cancer as set forth in claim 1, wherein the nicotinic acid derivative is selected from a group consisting of niacinamide, nicotinic acid, 6-aminonicotinamide, isonicotinamide, isonicotinohydrazide, N- methylnicotinamide, nicorandil, pyridine-2-carboxamide, thionicotin amide and 6-chloronicotinamide .

[Claim 6] The agent for the prevention and the treatment of cancer as set forth in claim 1, wherein the cancer is selected from a group consisting of stomach cancer, lung cancer, hepatoma, laryngeal cancer, colorectal cancer, bladder cancer, pancreatic cancer, pancreatic cancer, uterine cancer, esophageal cancer and breast cancer.

[Claim 7] A prevention and/or a treatment method for cancer including the step of administrating nicotinic acid derivative of claim 1 or pharmaceutically acceptable salts thereof to a target subject.

[Claim 8] The prevention and/or the treatment method for cancer as set fort in claim 7, wherein the nicotinic acid derivative is selected from a group consisting of niacinamide, nicotinic acid, 6-aminonicotinamide, isonicotinamide, isonicotinohydrazide, N- methylnicotinamide, nicorandil, pyridine-2-carboxamide, thionicotin amide and 6-chloronicotinamide .

[Claim 9] The prevention and/or the treatment method for cancer as set fort in claim 7, wherein the cancer is selected from a group consisting of stomach cancer, lung cancer, hepatoma, laryngeal cancer, colorectal cancer, bladder cancer, pancreatic cancer, pancreatic cancer, uterine cancer, esophageal cancer and breast cancer.

[Claim 10] A usage of nicotinic acid derivative of claim 1 or pharmaceutically acceptable salts thereof for the preparation of an agent for the prevention and/or the treatment of cancer.

[Claim ll] The usage as set forth in claim 10, wherein the nicotinic acid derivative is selected from a group consisting of niacinamide, nicotinic acid, 6- aminonicotinamide, isonicotinamide, isonicotinohydrazide, N-methylnicotinamide, nicorandil, pyridine-2-carboxamide, thionicotin amide and 6-chloronicotinamide .

[Claim 12] The usage as set forth in claim 10, wherein the cancer is selected from a group consisting of stomach cancer, lung cancer, hepatoma, laryngeal cancer, colorectal cancer, bladder cancer, pancreatic cancer, pancreatic cancer, uterine cancer, esophageal cancer and breast cancer.

[Claim 13] A method for the activation of RUNX3 or a gene coding RUNX3 including the step of administrating nicotinic acid derivative of claim 1 to a subject with cancer .

[Claim 14] The method as set forth in claim 13, wherein the subject with cancer has deacethylated RUNX3.

[Claim 15] The method as set forth in claim 13, wherein the subject with cancer has a methylated gene coding RUNX3.

[Claim 16] The method as set forth in claim 13, wherein the cancer is selected from a group consisting of stomach cancer, lung cancer, hepatoma, laryngeal cancer, colorectal cancer, bladder cancer, pancreatic cancer, pancreatic cancer, uterine cancer, esophageal cancer and breast cancer.