KR100710550B1 - Cancer cell-growth inhibition drugs comprising POH 1 gene or POH 1 protein, preparation method thereof and use thereof - Google Patents

Abstract

The present invention is a cancer cell proliferation inhibitor comprising a POH 1 protein or a POH 1 gene encoding one of the subunits of the reguratorey particles of the proteasome (proteasome) that degrades the protein as a post-transcriptional regulation in eukaryotic cells, It relates to a preparation method thereof and a method of inhibiting cancer cell proliferation using the same. According to the present invention, the cancer cell survival rate of up to 24% was obtained by treating the cancer cells with the inhibitor containing the POH 1 gene, and the mechanism of cancer cell proliferation through the POH 1 gene exhibiting an effective effect on inhibiting cancer cell proliferation. In addition to the overall understanding, it is expected to be useful for developing cancer diagnostic reagents and cancer treatments.

Proteasome, POH 1, Cancer Cells, Proliferation Inhibitor, Cancer Treatment

Description

Cancer cell growth inhibitor comprising POH1 gene or POH1 protein, preparation method thereof and method for inhibiting cancer cell proliferation using same {Cancer cell-growth inhibition drugs comprising POH 1 gene or POH 1 protein, preparation method

Figure 1 is a schematic diagram showing the pLNCX2 vector and subcloning process used in the present invention,

Figure 2 is a graph showing the colony forming assay (colony forming assay) results of the POH 1 gene introduced into the lung cancer cell line H1299 in accordance with the present invention, p53 gene,

Figure 3 is a colony forming (colony forming) picture formed after introducing the POH 1 gene represented by SEQ ID NO: 1 to cancer cells according to the present invention,

Figure 4a is a graph showing the results of the cell counting (cell counting assay) of POH 1 gene introduced into lung cancer cell line H1299 according to the present invention, and

Figure 4b is a graph showing the cell count analysis results of the POH 1 gene introduced into the lung cancer cell line H460 according to the present invention.

The present invention relates to a cancer cell proliferation inhibitor comprising a POH 1 gene or a POH 1 protein of a human similar to the Pad 1 gene of a yeast, a method for preparing the same, and a method of inhibiting cancer cell proliferation using the same, and more specifically, the POH The present invention relates to a cancer treatment method of inhibiting the proliferation of cancer cells by expressing one gene in cancer cells.

In general, cancer is known to occur due to abnormal proliferation and growth of cells due to various causes. These causes include exposure to chemical carcinogens, infection with carcinogenic viruses, and congenital genetic abnormalities. But fundamentally all of these causes cause abnormalities in intracellular genes.

Normal cells are complementarily regulated to grow and maintain the functions of oncogenes, tumor suppressor genes, and apoptosis regulating genes.

Cancer genes are normally essential genes for cell growth and differentiation, which contributes to cell proliferation and growth by promoting protein synthesis and intracellular signaling, but when mutations occur, they induce excessive cell proliferation. It is known to contribute.

Cancer is the world's second leading cause of death after circulatory disease, and research on cancer has been extensively performed. Currently known cancer treatments include surgery, radiation therapy, chemotherapy, immunotherapy, and gene therapy. There is this.

Meanwhile, as strategies for treating cancer are diversified, basic studies are being conducted to increase cancer survival by inhibiting cancer cell metastasis or inhibiting neovascularization, which is essential for cancer cell proliferation and metastasis. However, these treatment methods also have a high risk of side effects due to systemic administration of angiogenesis and metastasis inhibitors (chemical and protein preparations) as conventional chemotherapy involves side effects from systemic administration of anticancer drugs. In the course of the chemotherapy research, there has been a need for a new therapeutic method that selectively introduces and expresses genes for cancer treatment in cancer cells and cells in cancer tissues.

Therefore, the recent gene therapy method is to introduce long-term genes selectively into target cells to induce long-term expression, and to improve gene transfer efficiency when actually used in clinical treatment. Therefore, genes involved in cancer (cancer genes) And tumor suppressor genes) and genes involved in immune responses against cancer cells (cytokine genes) have been identified, suggesting that cancer is a disease that can be treated using various methods of gene therapy.

The methods used to introduce genes into cells can be divided into physical and chemical methods (Calcium-posphate coprecipitation, microinjection, electrophoresis, liposome), and biological methods (vector system), but they are still the most efficient and safe method of gene delivery. It is not possible to conclude which one, which is why a lot of research is focused on this, and now, a method of transferring genes by using a combination of physical, chemical and biological methods is widely used.

The prior art for gene therapy using the gene or its protein in the treatment of cancer is as follows.                         

Korean Patent Laid-Open No. 2001-112108 discloses a tumor suppressor using p43 protein as an active ingredient, which includes a specific base sequence that induces cytokines to increase an immune response and inhibit angiogenesis.

Korean Patent Laid-Open Publication No. 2002-40521 discloses an anticancer agent containing mycolactone, a substance that increases cancer cell death, an antisense Rb nucleotide and an anticancer agent containing mycolactone and mycolactone and the antisense Rb nucleotide, which decrease the expression of retinoblastoma protein, have.

US Pat. No. 6,329,505 discloses a composition comprising one or more prostate-specific proteins or genes encoding them for use in the treatment and diagnosis of prostate cancer.

In addition to the above patents, a gene or a protein thereof is often used for the treatment of cancer, and research on this is ongoing. Therefore, there is a demand for a gene that can be applied to the gene treatment of cancer more effectively.

Therefore, in the present invention, as a result of searching for various genes capable of inhibiting cancer cell proliferation, the POH 1 gene constituting the regulatory particle subunit of proteasome shows the effect of inhibiting the proliferation of cancer cells. The present invention was completed based on this.

Accordingly, it is an object of the present invention to provide a cancer cell proliferation inhibitor comprising a POH 1 gene or a POH 1 protein capable of inhibiting cancer cell proliferation.                         

Another object of the present invention is to provide a vector into which the POH 1 gene is introduced.

Another object of the present invention to provide a method for producing the cancer cell proliferation inhibitor.

Another object of the present invention to provide a method for inhibiting cancer cell proliferation using the inhibitor.

In order to achieve the above object, the cancer cell proliferation inhibitor of the present invention comprises a POH 1 gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or POH 1 protein represented by SEQ ID NO: 2.

In order to achieve the above another object, the vector into which the POH 1 gene of the present invention is introduced consists of introducing the POH 1 gene represented by SEQ ID NO: 1 into the pLNCX2 vector.

In order to achieve the above another object, the method for producing a cancer cell growth inhibitor of the present invention consists of introducing the POH 1 gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 into the pLNCX2 vector.

In order to achieve the above another object, the cancer cell proliferation inhibition method of the present invention is to administer the pLNCX2 vector containing the POH 1 gene 0.1 to 2㎛ / 10 ⁵ cells and liposomes 1 to 20㎍ / 10 ⁵ cells to cancer cells Is done.

Hereinafter, the present invention will be described in more detail.                     

As described above, the present invention relates to a cancer cell proliferation inhibitor comprising a POH 1 gene or a POH 1 protein, a preparation method thereof, and a method of inhibiting cancer cell proliferation using the same.

POH 1 protein constitutes ubiquitin-proteasome, an important proteolytic mechanism of post-transcriptional regulation in eukaryotic cells. The proteasome consists of a large multi-subunit enzyme complex covered at both ends with a regulatory particle (RP) and a regulatory moiety, the central area of the proteasome having a proteolytic activity area, and the regulatory part being a ubiquitinated protein. It recognizes and transfers to the proteolytic active region. On the other hand, the regulatory portion comprises at least 17 subunits having at least two structural and functional domains, and also a conserved region and ubiquitinated substrate having ATPase activity for unfolding a specific portion of the protein to be degraded. Have one or more different lids that appear to impart specificity for.

POH 1 protein, known as Pad 1 (in yeast), Rpn 11 Mrp 1, is one of the subunits of the regulatory region lead region, which is responsible for the positive modulator of transcription and multidrug resistance (MDR). It is also known that overexpression of Pad or recombinant POH 1 in Schizosaccharomyces pombe increases DNA binding activity leading to transactivation of AP-1 reactive genes (Shimanuki et al. al. J. Cell Sci. 1995, 108: 569-79 and Spataro et al. J. Biol. Chem. 1997, 272: 30470-8). On the other hand, recent studies have reported that Pad 1 motiles of Schistosoma mansoni stabilize c-Jun (Joseph F., et al. Molecular & Biochemical Parasitology 2002, 121: 163-172). ).

In the present invention, as described above, the effect of inhibiting cancer cell proliferation of POH 1 gene, which is one of the regulatory subunits of the proteasome, was confirmed, and a cancer cell proliferation inhibitor including POH 1 gene or POH 1 protein was prepared.

The POH 1 gene may be isolated from human tissue or synthesized according to a known DNA synthesis method. In the present invention, a POH 1 gene derived from Homo Sapiens of about 1132 bp represented by SEQ ID NO: 1 and a POH 1 protein represented by SEQ ID NO: 2 were used (Spataro et al. J. Biol. Chem. 1997, 272). : 30470-8, GenBank GI 5031980).

The cancer cell proliferation inhibitor according to the present invention was obtained by inserting a POH 1 gene into a pLNCX2 vector. In the present invention, although a pLNCX2 vector was used, various expression vectors are possible, so that other expression vectors can be easily used according to the purpose of expression. In addition, the size, nucleotide sequence, etc. of the gene inserted into the foreign gene insertion site of the expression vector can also be variously changed by known techniques.

The prepared vector is transduced into cancer cells in a conventional manner, and can be expressed with high efficiency to effectively inhibit cancer cell proliferation.

The concentration of the expression vector containing the POH 1 gene is administered to the cancer cells are not within the 0.1 2㎍ / 10 ⁵ cells, preferably from 1 to 2㎍ / 10 ⁵ cells, and liposomes with 1 to 20㎍ / 10 ⁵ cells It may be administered once or in several doses. However, the actual administration concentration of the active ingredient should be determined according to the type and characteristics of cancer cells, treatment conditions, administration methods and the like.

On the other hand, the inhibitor may be prepared in the form of an isotonic aqueous solution or suspension, and may contain an adjuvant such as a salt or buffer for controlling osmotic pressure, a hydrating agent, a stabilizer, and other therapeutically useful substances.

As described above, it was confirmed that POH 1, known as a protein constituting the regulatory portion of the proteasome, has the ability to inhibit cancer cell proliferation, which is compared with other gene therapy methods such as conventional tumor suppressor genes, radiation therapy or anticancer agent. The combination is expected to have a synergistic effect in the treatment of cancer.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Example 1

Cell culture

Human lung cancer cell lines (H460 and H1299) were purchased from the American Cell Line Bank (ATCC) and contained RPMI culture medium (GIBCO / BRL, Grand Island, USA) containing 10% fetal calf serum, 100 units / ml penicillin, and 100 μg / ml streptomycin. NY) was incubated at a temperature of 37 ℃ and 5% CO ₂ concentration conditions.

Example 2

Transfection

In inserting the POH 1 gene represented by SEQ ID NO: 1 into a pLNCX2 vector (Clontech Laboratories, Inc.), the plasmid containing the POH 1 gene represented by SEQ ID NO: 1 was digested with EcoRI / BamHI to ecoRI / BamHI of pcDNA3. After subcloning in, it was again cleaved with BamHI / NotI and subcloned with pLNCX2 to prepare a transfection vector containing the POH 1 gene, the process of which is shown in FIG. 1.

Comparative Example 1

Validation of cancer cell proliferation inhibitory effect using p53 gene

In order to establish optimal conditions for introducing POH 1 gene into cancer cells, anti-cancer gene p53, which is most widely used for gene therapy of various human cancers, was introduced into cancer cells and verified its inhibitory effect.

Cancer cells (1 × 10 ⁵ ) (H460 and H1299) were transferred to 60 mm culture dishes and these cells were incubated for 24 hours. After washing twice with serum free medium, the cells were placed in a mixture of 10 μg cationic liposomes (GIBCO / BRL, Grand Island, NY) and 2 μg pcDNA3 gene expression vector in 200 μl serum free medium and 200 μl serum free medium. A mixture of 10 μg of cationic liposomes (GIBCO / BRL, Grand Island, NY) and 2 μg of p53 gene expression vector were each incubated for 5 hours. Cells were then washed twice with serum free medium, then added to the culture medium and incubated for 16 hours. After further incubation at 37 ° C. for 24 hours, neomycin (GIBCO / BRL, Grand Island, NY) was treated at a concentration of 800 μg / ml, followed by incubation for 6 days. After staining the formed colonies with methylene blue, the proliferation change of cancer cells through colony forming assay (Table 1 and Figure 2) is shown.

Colonies () (%) LNCX2 plasmid 334 100 p53 gene 25 7.5

As can be seen from Table 1, when the p53 gene was introduced, the number of colonies was 25 (7.5%), and the cancer cell growth rate was significantly suppressed. Thus, it was confirmed that the tumor suppressor gene p53 was well introduced under the above conditions.

Example 3

Colony Formation Analysis

The POH 1 gene was introduced into cancer cells under the same conditions as in Comparative Example 1 except that the POH 1 gene and the LNCX2 vector were used. At this time, the amount of the expression vector containing POH 1 gene introduced into the cancer cells was changed to a concentration of 0.5 to 2.0 µg. .

Cancer cell proliferation inhibitory effect was confirmed through colony formation analysis and the results are shown in Table 2 and FIG. 3.

LNCX2  Concentration of POH 1-containing Expression Vector (㎍) 0.5 1.0 1.5 2.0 Colonies () 359 271 246 250 235 (%) 100 75.5 68.5 69.6 65.5

As can be seen from Table 2, the colony number of the expression vector containing the POH 1 gene was reduced to 60-70% compared to the control using only the LNCX2 plasmid.

Example 4

Cell Counting Assay

Cancer cells (1 × 10 ⁵ ) (H1299 and H460) were transferred to 35 mm culture dishes and these cells were incubated for 24 hours. After washing twice with serum free medium, the cells were mixed with 10 μg cationic liposomes (GIBCO / BRL, Grand Island, NY) and 1 μg LNCX2 plasmid in 100 μl serum free medium and cationic in 100 μl serum free medium. A mixture of 10 μg of liposomes (GIBCO / BRL, Grand Island, NY) and 0.1-2 μg of POH 1 gene expression vector pLNCX2 were each incubated for 5 hours. The cells were washed twice with serum free medium and then added to the culture medium and incubated for 16 hours. Three days after the transformation was repeated once more, cells were removed with trypsin and stained with 0.4% trypan blue to count the cells, and the results are shown in Table 3 and FIGS. 4A and 4B.

Expression vector concentration containing POH 1 gene (µg) Cell survival rate (%) LNCX2 plasmid Contains POH 1 gene H1299 0 100 100 0.1 102.2 59.5 0.5 117.6 46.4 One 119.8 32.1 2 86.9 23.8 H460 0  100 100 0.1 98.6 69.9 0.5 101.4 55.4 One 95.8 55.4 2 97.2 53.0

As can be seen from Table 3, when POH 1 was introduced into cancer cells, cell viability was reduced by 20 to 70% compared to the control group (LNCX2 plasmid), and in particular, expression vector 2 containing POH 1 gene in lung cancer cell line H1299. When ㎍ was introduced, the cell survival rate was 23.8%, which was very low.

As described above, the cancer cell proliferation inhibitor including the POH 1 gene according to the present invention was used to obtain an effective effect on suppressing cancer cell proliferation, and the overall understanding of the mechanism of cancer cell proliferation through the ability to inhibit cancer cell proliferation of the POH 1 gene, In combination with other gene therapies such as tumor suppressor genes, radiation therapy or anticancer agents, it is expected to have a synergistic effect in the treatment of cancer.

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Claims (6)

Cancer cell proliferation inhibitor comprising a POH 1 gene represented by SEQ ID NO: 1. Cancer cell proliferation inhibitor comprising a POH 1 protein represented by SEQ ID NO: 2. delete A method for producing a cancer cell proliferation inhibitor, characterized by introducing the POH 1 gene represented by SEQ ID NO: 1 into the pLNCX2 vector. delete delete

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