KR20040101707A - Fragment of FAF-1, and tumor metastasis inhibitory agents containing the same - Google Patents

Abstract

PURPOSE: Provided is a fragment of FAF-1(Fas-associated Factor 1) which has excellent inhibitory effects on angiogenesis and cell proliferation without cytotoxicity. Therefore, it is useful for an anticancer agent, especially, a tumor metastasis inhibitory agent. CONSTITUTION: The fraction having inhibitory effect on tumor metastasis is characteristically composed of 290-345 amino acids of FAF1(Fas-associated Factor 1). Its amino acid sequence is represented by the SEQ ID NO:1. Its coding DNA is represented by the SEQ IN NO:2. The anticancer agent is characterized by containing the fraction of FAF1.

Description

Fragment of FAF-1, and tumor metastasis inhibitory agents containing the same

The present invention relates to fragments of certain sites of FAF1 (Fas-associated Factor 1) and their use as anticancer agents, in particular tumor metastasis inhibitors. More specifically, the present invention is directed to fragments of amino acid 290-345 sites of FAF1, and to their use as anticancer agents, in particular tumor metastasis inhibitors, by inhibiting angiogenesis and tube formation, and cell nontoxic and proliferative inhibitory activity. It is about.

The life control phenomena caused by cell death appear well in the process of development of multicellular organisms, tissue differentiation, or diseases such as cancer, immunodeficiency, and degenerative diseases. In most cases, it is a specific form of apoptosis. Society, Genetics Review 2 (CH Cho ed) World Science Press, Korea, 1998, pp77-78).

The Fas (TNF: member of the tumor necrosis factor receptor superfamily) pathway, one of several pathways involved in apoptosis, is an essential pathway for normal development, and the genetic variation of Fas is an immunosuppressive disease. (Nagata, 1997). Recently, studies on the association between the Fas pathway and anticancer drugs, such as cisplatin or 5-FU (fluorouracil), and the Fas pathway, have been reported to be associated with cell death and cancer. (Fulda et al ., 1998; Friesen et al ., 1999; Houghton et al ., 1999; Watson, 1999).

Apoptosis by Fas signaling is known to be performed by a death domain (DD) consisting of about 80 amino acids in communication with signal transmitters (Nagata and Golstein, 1995; Nagata, 1997). In addition, FAP-1 (Fas associated phosphatase-1) (Yanagisawa et al. , 1997; Ryu et al., 1999), which performs its function without DD, has been reported.

Fas-induced apoptosis is substantially carried out by the activity of caspase, a type of cysteine protease in the lower-level signaling system, and many caspase substrates have been identified and used commercially as markers of apoptosis. have. In addition, the use of a conventional anticancer agent without artificial Fas signal has been reported to activate the caspase to enhance the susceptibility of cancer cell death (Wesselborg et al ., 1999). In particular, caspase-9 and caspase-8 are important in this process.

FAF1, on the other hand, is a protein associated with the Fas pathway, recently found in mice (Chu et al ., 1995; Becker et al ., 1997) and quails (Frohlich et al ., 1998), the first in humans. Has been isolated (Ryu et al ., 1999). They each have high amino acid homology, with 649 amino acids in mice, 648 amino acids in quails and 650 amino acids in humans.

The inventors have for the first time revealed that human FAF1 has the ability to induce apoptosis and that the minimal induction region is the 181-381 region of the FAF1 amino acid. Furthermore, the present inventors have found that FAF1 is a member of the Fas-death inducing signaling complex (Fas-DISC), and that DISC is the DED of caspase-8 and Fas-associated death domain protein (FADD). effector domain) and the amino acid 181-381 region of FAF1, which is structurally similar to DED, was identified as DEDID (DED-interacting domain) (amino acid and base sequence of FAF1-DEDID). 1a and 1b). Furthermore, when apoptosis was induced by treating Fas antibody (CH-11), an apoptosis inducer, apoptosis was inhibited by a deletion mutation of DEDID, and DEDID deletion mutations (FAF1-ΔDEDID) were associated with FAF1 protein function. It has been shown to act as a dominant negative (Ryu and Kim, 2003).

Now, we have a specific site, among the amino acid 181-381 sites (DEDID) of FAF1, which can be usefully used as anticancer agents, especially tumor metastasis inhibitors, by having specific angiogenesis and tube formation inhibition, and cell nontoxic and proliferation inhibitory activity. A new fragment of was found.

It is therefore an object of the present invention to provide new FAF1 fragments with inhibitory angiogenesis and tube formation and cell nontoxic and proliferative inhibitory activity.

Another object of the invention is to provide the use of said FAF1 fragment as an anticancer agent, in particular as a tumor metastasis inhibitor.

1 is a diagram showing an amino acid sequence (1a) and a nucleotide sequence (1b) of a DEDID (181-381) of FAF1;

2 shows amino acid sequence (2a) and nucleotide sequence (2b) of FAF1 fragment (290-345);

3 is a photograph showing the angiogenesis inhibitory activity of FAF1 fragments 290-345;

4 is a photograph showing tube forming inhibitory activity of FAF1 fragments 290-345;

FIG. 5 shows the results of confirming the cell nontoxicity of FAF1 fragments 290-345 by LDH release assay; FIG.

Figure 6 is a graph showing the results confirmed by XTT analysis of cell proliferation inhibitory activity of FAF1 fragment (290-345).

First, the present invention relates to fragments of the amino acid 290-345 region of FAF1 having tumor metastasis inhibiting activity, in particular fragments having the amino acid sequence of SEQ ID NO: 1.

Secondly, the present invention relates to DNA encoding the FAF1 fragment, in particular DNA having the nucleotide sequence of SEQ ID NO: 2.

Third, the present invention relates to a recombinant vector containing the DNA.

Fourth, the present invention relates to a bacterial cell transformed with the recombinant vector.

Fifth, the present invention relates to a method for preparing a FAF1 fragment, comprising culturing the bacterial cells under conditions capable of expressing the FAF1 fragment, and recovering the FAF1 fragment expressed from the culture.

Sixth, the present invention relates to an anticancer agent, particularly a tumor metastasis inhibitor, containing the FAF1 fragment as an active ingredient.

Hereinafter, the present invention will be described in detail.

The inventors have found that the amino acid 181-381 site (DEDID) of FAF1, which has been identified by the inventors as a member of Fas-DISC to interact with caspase-8 and DED of FADD, is used as an anticancer agent, in particular a tumor metastasis inhibitor I thought that I could. Thus, to find sites with tumor metastasis suppression ability, several fragments of FAF1-DEDID (e.g., FAF1 (181-212), FAF1 (212-290), FAF1 (290-381), FAF1 (290-345)) ) And their ability to inhibit tumor metastasis.

To assess the ability of FAF1 fragments to inhibit tumor metastasis, a chorioallantoic membrane (CAM) assay and a tube formation assay of human umbilical vein endothelial cells (HUVEC) cells were first used to assess angiogenesis. Used. In addition, in order to measure the cytotoxicity and cell proliferation inhibitory activity of FAF1, lactate dehydrogenase (LDH) release assay and XTT (bis- (2-methoxy-4-nitro-5-sulphenyl)-(2H) -tetrazolium-5 -carboxanilide) analysis was used.

As a result, it was surprisingly confirmed that the amino acid 290-345 region included in the DEDID of FAF1, which was found by the present inventors, effectively inhibited angiogenesis and tube formation, and was nontoxic to cells and had proliferation inhibitory activity. Therefore, amino acid 290-345 region of FAF1 of the present invention can be usefully used as an anticancer agent, especially a tumor metastasis inhibitor.

FAF1 fragments according to the invention can be chemically synthesized or prepared using genetic recombination techniques. For example, FAF1 fragments of the invention can be prepared by recombinant DNA techniques using expression vectors into which DNA having a base sequence encoding it is inserted. The vector is prepared to be transformed into a suitable host cell and expressed under suitable conditions according to the method of Sambrook et al. (Molecular Cloning, 1989, Cold Spring Harbor Laboratory Press). It may also be prepared using a fusion protein comprising an amino acid sequence according to the present invention.

On the other hand, the amino acid sequence of the FAF1 fragment according to the present invention can be modified constantly, for example, by increasing or decreasing the number of amino acids. It may also be modified by changing the specific residue components or the order thereof except within the range that does not reduce the activity of the FAF1 fragment according to the invention, the residues that must be directly involved in or conserved in binding. Modifiable amino acids can be modified not only with naturally occurring L-α-amino acids, but also with β, γ, δ amino acids as well as derivatives of D-α-amino acids.

Typically, investigating the effect of electrostatic force or hydrophilicity on binding using a peptide substituted with one amino acid, the sensitivity of the positively charged amino acids (eg Lys, Arg) or negatively charged amino acids (eg Glu) It can be seen that. As such, the number or type of residues to be substituted or added is determined by the required space, such as the required spacing and hydrophilicity or hydrophobicity between the necessary points of attachment.

Substitutions often result in significant changes in function. The choice of residues to be altered can also have a major impact on maintaining the basic backbone of the peptide, such as the electrical conductivity, hydrophobicity, side chain changes, or helix structure of the molecules present at the desired position. In general, significant changes in the properties of peptides include those in which hydrophilic residues such as serine are replaced by hydrophobic residues such as leucine, isoleucine, phenylalanine, valine or alanine, or electrically positive residues such as lysine, arginine or histidine. This is the case when an amino acid having no side chain such as glycine is substituted with an electrically negative residue such as glutamic acid or aspartic acid, or a residue having a bulky side chain.

In view of the above facts, a person of ordinary skill in the art uses conventional techniques within the range that does not maintain, increase or impair the ability of the amino acid sequence set forth in SEQ ID NO: 1 to inhibit its tumor metastasis. Can be modified, and this is also within the scope of the present invention.

The total daily dose to be administered to a patient when administered for clinical purposes is in the range of 0.05-200 mg / kg body weight, preferably 0.05 mg / kg body weight. However, the dosage for a particular patient may be increased or decreased depending on the weight, sex, health condition, diet, time of administration, method of administration, and severity of the disease .

FAF1 fragments of the present invention can be directly injected or formulated in solution or micelle form. The anticancer agent according to the present invention, in particular the tumor metastasis inhibitor, may be applied to the human body by parenteral or topical administration, preferably by intravenous injection, subcutaneous injection, endothelial injection, intramuscular injection, or the like. For this purpose, the active ingredient of the invention can be suspended or dissolved in a pharmaceutically acceptable carrier, for example a water soluble carrier.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended to help the understanding of the present invention, but are not intended to limit the scope of the present invention in any way.

Example 1: Preparation of GST-FAF1 (181-212), GST-FAF1 (212-290), GST-FAF1 (290-381) and GST-FAF1 (290-345)

In the present invention, GST (glutathion-S-transferase) -tagged by dividing FAF1-DEDID (having the amino acid sequence of SEQ ID NO: 3), which is found to interact with caspase-8 and DED of FADD as a member of Fas-DISC The vector was cloned into the pGEX 4T-1 vector (Pharmacia Biotech, Uppsala, Sweden). To clone FAF1 deletion mutants into GST fusion vectors, PCR was performed using GST-FAF1 full length cDNA as a template with primers comprising Eco RI and Xho I linkers. The recombinant vector was transformed into E. coli BL21 (DE3) strain to obtain transformed colonies. To isolate the GST fusion protein, cells were incubated at 37 ° C. in a medium with an OD ₆₀₀ of 0.5-0.6, and then GST-FAF1-deleted by adding IPTG (isopropyl-β-D-galactopyranoside) at a concentration of 0.5 mM to the medium. Induced expression of mutants. Cells were then sonicated in ice-cold lysis buffer (200 mM Tris-Cl, pH 8.0, 0.5 M NaCl, 100 μM EDTA, 0.1% Triton X-100, 0.4 mM PMSF), followed by centrifugation at 13,000 rpm for 10 minutes, only the supernatant. Collected. 20 μl of glutathione-agarose beads (Peptron, South Korea) was added to 200 μl of the supernatant, incubated at 4 ° C. for 4 hours, and the resulting GST-fusion protein was electrophoresed on an SDS-PAGE gel, followed by coomassie staining. Normalized through.

Example 2: Determination of Angiogenesis Inhibitory Activity of GST-Fusion Proteins

As part of evaluating the tumor metastasis inhibiting ability of the GST-fusion protein prepared in Example 1, CAM assay was first used to measure their angiogenesis inhibitory activity.

For analysis, chicken eggs were incubated for 3 days in a thermohygrostat at a temperature of at least 70% and a temperature of 37 ° C., and albumin was extracted with 2-3 G with a 26 G (gauge) syringe. After sealing with transparent tape to prevent evaporation of water, a window about 1 × 1 cm in size was made with a drill in the center of the fertilized egg. 10 μg of GST-fusion protein was dissolved in 10 μl of distilled water, dropped onto a thermoox disc, dried, and placed on the villus ureum exposed through the window. It was sealed with a transparent tape and incubated for 3 days in a 37 DEG C thermostat. In order to distinguish the blood vessels distributed in the ureter cavity, intralipid was injected into the ureter cavity using a 26 G syringe to cover the blood vessels distributed in the ureter cavity, and then the vascular changes distributed in the chorionic ureter were compared. The results are shown in FIG. As shown in Figure 3, it was confirmed that capillary neovascularization of the site treated with GST-FAF1 (290-345) is effectively inhibited compared to other sites.

Example 3 Determination of Pipe Formation Inhibitory Activity of GST-Fusion Proteins

As part of evaluating the tumor metastasis inhibiting ability of the GST-fusion protein prepared in Example 1, the degree of tube formation of HUVEC cells, which are human placental cell lines, was analyzed.

For analysis, 50 μl of growth factor-free Matrigel (10 mg / ml) was added to a 96 well plate and polymerized at 37 ° C. for 1 hour. About 10 ⁵ HUVEC cells were plated here and treated with GST-fusion protein after 1-2 hours. After 24 hours, the degree of tube formation was observed through a microscope. The results are shown in FIG. As shown in FIG. 4, it was confirmed that tube formation was effectively inhibited by amino acid 290-345 sites of FAF1.

Example 4: Determination of cell nontoxic and proliferation inhibitory activity of FAF1 (290-345) protein

In order to investigate whether the angiogenesis and tube formation inhibitory activity of the FAF1 fragment (290-345) identified in the above example was due to cytotoxicity, LDH release assay using HUVEC cells was used, and the results are shown in FIG. 5. Indicated. In addition, in order to measure the cell proliferation inhibitory activity of the FAF1 fragment (290-345), XTT assay was used, the results are shown in FIG.

As shown in Figure 5 and 6, the addition of FAF1 fragment (290-345) was not confirmed the cell killing (necrosis), it was confirmed that only proliferation of the cell significantly reduced compared to the control.

As confirmed above, fragments of the 290-345 site in the DEDID of FAF1 have excellent antiangiogenic and vascular formation inhibition, and cell nontoxic and proliferative inhibitory activity, and thus can be usefully used as anticancer agents, particularly tumor metastasis inhibitors.

<110> Pai Chai University <120> Fragment of FAF1, and tumor metastasis inhibitory agents          containing the same <130> PC03-0156-EHK <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 56 <212> PRT <213> Homo sapiens <400> 1 Asp Ser Asp Gly Asp Asp Phe Glu Asp Ala Thr Glu Phe Gly Val Asp   1 5 10 15 Asp Gly Glu Val Phe Gly Met Ala Ser Ser Ala Leu Arg Lys Ser Pro              20 25 30 Met Met Pro Glu Asn Ala Glu Asn Glu Gly Asp Ala Leu Leu Gln Phe          35 40 45 Thr Ala Glu Phe Ser Ser Arg Tyr      50 55 <210> 2 <211> 168 <212> DNA <213> Homo sapiens <400> 2 gatagcgatg gagatgactt tgaagatgct acagaatttg gggtggatga tggagaagta 60 tttggcatgg cgtcatctgc cttgagaaaa tctccaatga tgccagaaaa cgcagaaaat 120 gaaggagatg ccttattaca atttacagca gagttttctt caagatat 168 <210> 3 <211> 201 <212> PRT <213> Homo sapiens <400> 3 Ser His Ala Gly Ala Leu Gln Glu Ser Leu Asn Gln Asn Phe Met Leu   1 5 10 15 Ile Ile Thr His Arg Glu Val Gln Arg Glu Tyr Asn Leu Asn Phe Ser              20 25 30 Gly Ser Ser Thr Ile Gln Glu Val Lys Arg Asn Val Tyr Asp Leu Thr          35 40 45 Ser Ile Pro Val Arg His Gln Leu Trp Glu Gly Trp Pro Thr Ser Ala      50 55 60 Thr Asp Asp Ser Met Cys Leu Ala Glu Ser Gly Leu Ser Tyr Pro Cys  65 70 75 80 His Arg Leu Thr Val Gly Arg Arg Ser Ser Pro Ala Gln Thr Arg Glu                  85 90 95 Gln Ser Glu Glu Gln Ile Thr Asp Val His Met Val Ser Asp Ser Asp             100 105 110 Gly Asp Asp Phe Glu Asp Ala Thr Glu Phe Gly Val Asp Asp Gly Glu         115 120 125 Val Phe Gly Met Ala Ser Ser Ala Leu Arg Lys Ser Pro Met Met Pro     130 135 140 Glu Asn Ala Glu Asn Glu Gly Asp Ala Leu Leu Gln Phe Thr Ala Glu 145 150 155 160 Phe Ser Ser Arg Tyr Gly Asp Cys His Pro Val Phe Phe Ile Gly Ser                 165 170 175 Leu Glu Ala Ala Phe Gln Glu Ala Phe Tyr Val Lys Ala Arg Asp Arg             180 185 190 Lys Leu Leu Ala Ile Tyr Leu His His         195 200 <210> 4 <211> 603 <212> DNA <213> Homo sapiens <400> 4 agtcatgctg gtgccctgca ggagtcatta aatcaaaact tcatgctgat catcacccac 60 cgagaagtcc agcgggagta caacctgaac ttctcaggaa gcagtactat tcaagaggta 120 aagagaaatg tgtatgacct tacaagtatc cccgttcgcc accaattatg ggagggctgg 180 ccaacttctg ctacagacga ctcaatgtgt cttgctgaat cagggctctc ttatccctgc 240 catcgactta cagtgggaag aagatcttca cctgcacaga cccgggaaca gtcggaagaa 300 caaatcaccg atgttcatat ggttagtgat agcgatggag atgactttga agatgctaca 360 gaatttgggg tggatgatgg agaagtattt ggcatggcgt catctgcctt gagaaaatct 420 ccaatgatgc cagaaaacgc agaaaatgaa ggagatgcct tattacaatt tacagcagag 480 ttttcttcaa gatatggtga ttgccatcct gtatttttta ttggctcatt agaagctgct 540 tttcaagagg ccttctatgt gaaagcccga gatagaaagc ttcttgctat ctacctccac 600 cat 603

Claims (9)

A fragment of amino acid 290-345 region of FAF1 (Fas-associated Factor 1) having tumor metastasis inhibiting activity. The fragment of claim 1 having the amino acid sequence of SEQ ID NO: 1. DNA encoding the FAF1 fragment according to claim 1. The DNA of claim 3 having the nucleotide sequence of SEQ ID NO: 2. 5. A recombinant vector containing the DNA according to claim 3. A bacterial cell transformed with the recombinant vector according to claim 5. Claim 1 or claim 1 comprising the step of culturing the bacterial cell according to claim 6 under conditions capable of expressing the FAF1 fragment according to claim 1, and recovering the expressed FAF1 fragment from the culture. Method for preparing a FAF1 fragment according to claim 2. An anticancer agent containing the FAF1 fragment according to claim 1 as an active ingredient. The anticancer agent of claim 8 which is a tumor metastasis inhibitor.