WO2006016574A1

WO2006016574A1 - ANTINEOPLASTIC AGENT APPLYING RNAi

Info

Publication number: WO2006016574A1
Application number: PCT/JP2005/014566
Authority: WO
Inventors: Yasuharu Nishimura; Tetsuya Nakatsura; Seiji Hosaka
Original assignee: Kumamoto University
Priority date: 2004-08-12
Filing date: 2005-08-09
Publication date: 2006-02-16
Also published as: JPWO2006016574A1

Abstract

Disclosed is a novel antineoplastic agent applying RNAi phenomenon. Specifically disclosed is an antineoplastic agent containing a factor which is capable of suppressing expression of HSP105 through RNAi.

Description

Antitumor agents using RNAi

Technical field

[0001] The present invention relates to an antitumor agent using RNAi. More specifically, the present invention relates to an antitumor agent using a factor capable of suppressing the expression of HSP105 by RNAi.

Background art

[0002] RNAi functions are inhibited! / A double-stranded RNA consisting of sense RNA and antisense RNA that is homologous to a specific region of a gene interferes with the homologous portion of the target gene transcript mRNA. It was first proposed in 1998 by an experiment using nematodes. Later in 2001, it was found that 21-23 base pair double stranded RNA can induce RNAi effect in mammalian cells without cytotoxicity.

[0003] Conventional methods for analyzing gene function include methods such as homologous recombination gene knockout method, antisense method, and ribozyme method as methods for suppressing the function of genes that have been divided by conventional nucleotide sequences. Compared with RNA interference method, RNA interference method is simple and inexpensive, has the advantage that gene function can be analyzed in a short time, and the gene sequence to be inhibited should have at least 19 bases. It is. In addition, siRNA expression vectors have been developed, and delivery methods using viral vectors are also in the development stage for medical applications. As described above, the RNA interference method is used as a technique for specifically suppressing the synthesis of gene products, and its application to medicine has been reported, but there are few reports that it has actually been effective.

[0004] HSP105 is a heat shock protein belonging to the HSP105 / HSP110 family, and it is known that its expression is induced by various stresses such as heat shock and other drugs. Although its function has not yet been fully elucidated, it is thought to bind to various proteins and to be involved in the activity of protecting against degradation and suppressing function. In mouse embryos, it has been shown that expression is transiently increased during embryonic period (Hatayama. T. et al. Cell Struct. Funct. 22,517-525. (1997)) ₀ rat neurons It has also been shown that overexpression of HSP105 suppresses apoptosis induced by various stresses (Hatayama. T Et al. Biochem. Biophys. Res. Commun. 288, 528-534. (2001)). The present inventors have identified the ability to identify HSP105 in the SEREX method using serum from patients with spleen cancer and colon cancer. This indicates that IgG antibodies against HSP105 exist in the serum of cancer patients! (Nakatsura. T. et al. Biochem. Biophys. Res. Commun. 281, 936-944. (2001)). The present inventors have also found that HSP105 is overexpressed in various human tumors as well as spleen and colon cancers (Kai. M. et al. Oncol. Rep. 10.1777- 1782. (2003)). On the other hand, it was found that in normal human tissues, expression was highest in the testis and weak expression was observed in some organs such as the brain.

Disclosure of the invention

Problems to be solved by the invention

[0005] For cancer, multidisciplinary treatment using various methods has been performed as a treatment method for patients with difficult surgery or postoperative treatment, but there is still no definitive treatment method including the problem of side effects. The current situation is that it has not been established. An object of the present invention is to provide a novel antitumor agent utilizing the RNAi phenomenon. More specifically, an object of the present invention is to suppress the expression of HSP105 using siRNA of HSP105, study the effect on tumor, and provide a novel antitumor agent.

Means for solving the problem

[0006] Since the present inventors transformed the mouse NIH3T3 cells by introducing the HSP105 gene, the present inventors thought that HSP105 has an oncogene-like function, and the main one is to suppress apoptosis. Based on the above hypothesis, as a result of suppressing the expression of HSP105 in various cancers expressing HSP105 using the siRNA of HSP105, the present inventors succeeded in causing the cells to undergo apoptosis. The effect was more prominent in the cancer cells expressing wild-type p53, and the effect was increased when used in combination with the pile cancer agent adriamycin.

[0007] In view of the above results, gene therapy using the introduction of wild-type p53 gene into cancer cells to apoptosis has already been performed, but wild-type p53 is considered to be difficult to use. HSP105 siRNA is considered to be effective against cancers that express phenotypes, and in addition, this gene therapy also has a synergistic effect on cancers with mutant P53. I can expect.

[0008] That is, according to the present invention, there is provided an antitumor agent comprising a factor capable of suppressing the expression of HSP105 by RNAi.

According to another aspect of the present invention, there is provided an apoptosis inducer in cancer cells, comprising a factor capable of suppressing the expression of HSP105 by RNAi.

[0009] Preferably, the factor capable of suppressing the expression of HSP105 by RNAi is siRNA or shRNA.

Preferably, the agent of the present invention is used in combination with an anticancer agent.

Preferably, the agents of the present invention are used against cancer cells that express wild-type p53.

[0010] Preferably, the siRNA is a double-stranded RNA consisting of an RNA having the base sequence set forth in SEQ ID NO: 1 and an RNA having the base sequence set forth in SEQ ID NO: 2.

Preferably, the agent of the present invention is used for the treatment of colorectal cancer, knee cancer, esophageal cancer, breast cancer, malignant melanoma and the like that highly express HSP105.

[0011] According to yet another aspect of the present invention, there is provided a double-stranded RNA comprising RNA having the base sequence set forth in SEQ ID NO: 1 and RNA having the base sequence set forth in SEQ ID NO: 2.

[0012] According to still another aspect of the present invention, there is provided a method for suppressing a tumor, comprising administering a factor capable of suppressing the expression of HSP105 by RNAi to a mammal including a human.

According to still another aspect of the present invention, there is provided a method for inducing apoptosis in cancer cells, comprising administering a factor capable of suppressing the expression of HSP105 by RNAi to a mammal including a human.

[0013] According to still another aspect of the present invention, there is provided use of a factor capable of suppressing the expression of HSP105 by RNAi for the production of an antitumor agent.

According to yet another aspect of the present invention, there is provided use of a factor capable of suppressing the expression of HSP105 by RNAi for the production of an apoptosis inducer in cancer cells.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail. The present invention relates to an antitumor agent and an apoptosis-inducing agent in cancer cells (hereinafter, these may be collectively referred to as the drug of the present invention) containing a factor capable of suppressing the expression of HSP105 by RNAi. . Specific examples of factors that can suppress the expression of HSP105 by RNAi include siRNA and shRNA as described below. When such a factor is introduced into a cell, an RNAi phenomenon occurs and RNA having a homologous sequence is degraded. Such RNAi phenomenon is a phenomenon observed in nematodes, insects, protozoa, hydra, plants, vertebrates (including mammals).

[0015] According to a preferred embodiment, double-stranded RNA having a length of about 20 bases (for example, about 21 to 23 bases) or less, called siRNA, can be used in the present invention. Such siRNA can suppress gene expression by being expressed in cells, and can suppress expression of a target gene of the siRNA (in the present invention, HSP105 gene).

[0016] The siRNA used in the present invention may be in any form as long as it can cause RNAi. Here, “siRNA” is an abbreviation for short interfering RNA, which is artificially chemically synthesized, biochemically synthesized, or synthesized in organisms, or about This is short double-stranded RNA of 10 base pairs or more, which is formed by decomposing double-stranded RNA of 40 bases or more in the body, and usually has a 5'-phosphate or 3'-OH structure. The 3 'end protrudes about 2 bases. A specific protein binds to this siRNA, and RISC (RNA-induced-silencing-complex) is established. This complex recognizes and binds to mRNA having the same sequence as that of siRNA, and cleaves mRNA at the center of siRNA by RNaselll-like enzyme activity.

[0017] It is preferable that the siRNA sequence and the mRNA sequence to be cleaved as a target match 100%. However, when the base at the position outside the central force of the siRNA does not match, the cleavage activity by RNAi often remains partially, so it does not necessarily have to match 100%.

[0018] The region having homology between the nucleotide sequence of siRNA and the nucleotide sequence of HSP105 gene whose expression should be suppressed preferably does not include the translation initiation region of HSP105 gene. Since various transcription factors and translation factors are expected to bind to the translation initiation region, siRNA This is because it cannot be effectively bound to mRNA and the effect is expected to be reduced. Therefore, the homologous sequence is preferably 20 bases away from the translation start region of the HSP105 gene! /, More preferably 70 bases away from the translation start region of the HSP105 gene. The sequence having homology may be, for example, a sequence near the 3 ′ end of the HSP105 gene.

[0019] In the present invention, siRNA can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, dsRNA of about 40 bases or more) can be used as such a factor. For example, at least about 70%, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more of a part of the nucleic acid sequence of the HSP105 gene. RNA containing a double-stranded portion or a variant thereof containing a sequence having a homology of at least%, most preferably 100%, can be used. The sequence portion having homology is usually at least about 15 nucleotides or more, preferably at least about 19 nucleotides, more preferably at least about 20 nucleotides in length, and even more preferably at least about 21 nucleotides in length. .

[0020] Specific examples of siRNA that can be used in the present invention include RNA having a 21-base base sequence corresponding to the 138th to 158th positions of HSP105 mRNA, specifically, SEQ ID NO: 1 Forces including, for example, RNA having the nucleotide sequence described and RNA having the nucleotide sequence set forth in SEQ ID NO: 2 are not limited thereto. The base sequence of the HSP105 gene is known, and is described in, for example, NCBI, Nucleotide Sequence Data base accession No. AB003334.

[0021] According to another aspect of the present invention, as a factor capable of suppressing the expression of HSP105 by RNAi, a shRNA (short hair pin RNA) comprising a short hairpin structure having a protruding portion at the 3 'end Can be used. A shRNA is a molecule of about 20 base pairs or more that has a double-stranded structure within the molecule and has a hairpin-like structure by including a partially palindromic base sequence with single-stranded RNA. Say. After such shRNA is introduced into the cell, it is decomposed into a length of about 20 bases (typically, for example, 21 bases, 22 bases, 23 bases) in the cell, and RNAi can be digested in the same manner as siRNA. Can cause. As mentioned above, shRNA causes RNAi as well as siRNA, and can be used effectively in the present invention. The

[0022] The shRNA preferably has a 3 'protruding end. The length of the double-stranded part is not particularly limited, but is preferably about 10 nucleotides or more, more preferably about 20 nucleotides or more. Here, the 3 ′ protruding end is preferably DNA, more preferably DNA of at least 2 nucleotides, and further preferably DNA of 2 to 4 nucleotides.

[0023] As described above, in the present invention, siRNA or shRNA can be used as a factor capable of suppressing the expression of HSP105 by RNAi. The advantages of siRNA are as follows: (1) Even if it is introduced into cells, RNA itself is not integrated into the chromosome of normal cells! /, so it is safer than treatment that causes mutations transmitted to offspring, And (2) short double-stranded RNA is relatively easy to chemically synthesize and is more stable when double-stranded. In addition, as an advantage of shRNA, when treatment is performed by suppressing gene expression for a long time, a vector that transcribes shRNA in a cell can be prepared and introduced into the cell.

[0024] The factor capable of suppressing the expression of HSP105 by RNAi used in the present invention (that is, siRNA or shRNA as described above) may be artificially chemically synthesized, sense strand and antisense. It can also be prepared by synthesizing RNA in vitro with T7 RNA polymerase, which has a hairpin structure in which the DNA sequences of the strands are ligated in the opposite direction. When synthesized in vitro, antisense and sense RNAs can be synthesized from the truncated DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is triggered and HSP1 05 expression is suppressed. Here, such RNA can be introduced into cells using, for example, the calcium phosphate method or various transfection reagents (eg, oligofectamine, Lipofectamine, lipofection, etc.).

[0025] Furthermore, according to the present invention, there is provided an expression vector comprising a nucleic acid sequence encoding a factor (preferably siRNA or shRNA) capable of suppressing the expression of HSP105 by RNAi. Furthermore, according to the present invention, a cell containing the above-described expression vector is provided. The cells of the present invention may transiently or stably express a factor that causes RNAi. The expression vectors and cell types described above are not particularly limited, but are preferably used for treatment. It can be done.

[0026] The agent of the present invention can be widely used for tumor suppression. More specifically, tumor suppression includes prevention of tumor development, suppression of tumor growth, tumor regression, and suppression of tumor migration. Clinically, cancer and Z or tumor prevention and Z Or means to encompass all of the treatment.

[0027] The types of cancer for which the antitumor agent of the present invention can be used are not particularly limited, and include all of benign tumors and malignant tumors. Specific examples of cancer include malignant melanoma, malignant lymphoma, digestive cancer, lung cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, colon cancer, ureteral tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, breast cancer, Liver cancer, spleen cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, ovarian cancer, uterine cancer, prostate cancer, thyroid cancer, brain tumor, force positive sarcoma, hemangioma, leukemia , Polycythemia vera, neuroblastoma, retinoblastoma, myeloma, cystoma, sarcoma, osteosarcoma, myoma, skin cancer, basal cell cancer, skin appendage cancer, skin metastasis cancer, skin melanoma, etc. It is not limited to these.

[0028] The agent of the present invention can be used in combination with other anticancer agents. In such a case, the agent of the present invention can be administered together with certain anticancer agents or separately before and after. By administering another anticancer agent in this manner, cancer cells that have been resistant to such an anticancer agent can be effectively treated or prevented.

[0029] Specific examples of the anticancer agent that can be used in combination with the drug of the present invention include, but are not limited to, the following.

(1) Alkylating agents (for example, cyclophosphamide, busulfan, thiotepa, dacarbazine, etc.)

(2) Antimetabolite (eg, methotrexate, 6-mercaptopurine, fluorouracil (5 FU), etc.)

(3) DNA topoisomerase inhibitors (for example, camptothecin, etoposide, etc.)

(4) Tubulin agonists (vinblastine, vincristine, etc.)

(5) Platinum compounds (cisbratine, carbobratin, etc.)

(6) Anticancer antibiotics (adriamycin, daunorubicin, mitomycin C, bleomycin, etc.) (7) Hormonal agents (tamoxifen, leuprorelin, etc.)

(8) Biologics (asparaginase, interferon, etc.)

(9) Immunostimulants (such as lentinans from Shitake)

[0030] The administration method of the drug of the present invention includes oral administration and parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, administration to the affected area. Topical administration, dermal administration, etc.) and direct administration to the affected area.

[0031] When the agent of the present invention is used as a pharmaceutical composition, a pharmaceutically acceptable additive can be blended as necessary. Specific examples of pharmaceutically acceptable additives include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffering agents, Powers including, but not limited to, delivery vehicles, diluents, carriers, excipients and / or pharmaceutical adjuvants.

[0032] The dosage form of the drug of the present invention is not particularly limited! Examples of sputum include liquids, injections, sustained-release agents and the like. The solvent used for formulating the drug of the present invention as the above-mentioned preparation may be either aqueous or non-aqueous.

[0033] Injections can be prepared by methods well known in the art. For example, after being dissolved in a suitable solvent (saline, buffer solution such as PBS, sterilized water, etc.), sterilized by filtration with a filter or the like, and then filled into a sterile container (for example, an ampule). Can be prepared. This injection may contain a conventional pharmaceutical carrier, if necessary. Administration methods using non-invasive catheters can also be used. Examples of the carrier that can be used in the present invention include neutral buffered physiological saline or physiological saline mixed with serum albumin.

[0034] Furthermore, the factor capable of suppressing the expression of HSP105 by RNAi, which is an active ingredient of the drug of the present invention, can be administered in the form of a non-viral vector or a viral vector. Such administration forms are known in the art, for example, a separate volume of experimental medicine “Basic technology of gene therapy” Yodosha, 1996; a separate volume of experimental medicine “Gene transfer & expression analysis experiment method” Yodosha, It is described in 1997.

[0035] In the case of a non-viral vector form, a method of introducing a nucleic acid molecule using ribosome (liposome method, HVJ-ribosome method, catonic ribosome method, lipofussion method, ribosome Etatamine method, etc.), microinjection method, and method of transferring nucleic acid molecules to cells together with carriers (metal particles) using a gene gun. The originating expression vector, for _{example, P CAGGS, pBJ- CMV, pcDNA3} . 1, pZeoSV (lnv itrogen Company or Stratagene Inc. force available), and the like.

[0036] When using lipofusion, for example, lipofectamine 2000, oligofectamine

(.silencer siRNA TransfectionKit.ueneSilencer siRNA Transfection Reagent)!

[0037] The HVJ-ribosome method encapsulates a nucleic acid molecule in a ribosome made of a lipid bilayer, and fuses the ribosome with an inactivated Sendai Winores (Hemagglutinating virus of Japan, HVJ). Include. The HVJ-ribosome preparation method is described, for example, in a separate volume of experimental medicine “Basic Technology of Gene Therapy” Yodosha, 1996; a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experiment Method” Yodosha, 1997.

[0038] When a factor capable of suppressing the expression of HSP105 by RNAi is administered to a living body using a viral vector, a viral vector such as a recombinant adenovirus or a retrovirus can be used. Detoxified retroviruses, adenoviruses, adeno-associated winoles, henorepesuinores, vaccinia winoles, box winenores, poliowinoles, sindbiswinores, sendai virus, SV40, and other DNA viruses or RNA viruses, RNAi HSP105 A gene can be introduced into a cell or tissue by introducing a DNA that expresses a factor capable of suppressing the expression of the gene and infecting the cell or tissue with the recombinant virus.

[0039] Furthermore, a factor capable of suppressing the expression of HSP105 by RNAi can be directly injected into a living organ or tissue.

[0040] The dosage of the drug of the present invention is a factor that can suppress the expression of HSP105 by the purpose of use, the severity of the disease, the patient's age, weight, sex, medical history, or RNAi as an active ingredient. It can be determined by those skilled in the art in consideration of the type of the above.

[0041] The dose of the factor capable of suppressing the expression of HSP105 by RNAi as an active ingredient is not particularly limited, and is, for example, about 0.1 ng to about 100 mgZkg, preferably about 1 ng to about 10 mg. When administered as a viral or non-viral vector, Per adult, it is usually from 0.0001 to 100 mg, preferably from 0.001 to 10 mg, more preferably from 0.01 to Lmg.

[0042] The frequency of administration of the drug of the present invention is, for example, once a day to once every several months (for example,

, Once a week to once a month). RNAi is generally effective for 1-3 days after administration. Therefore, it is preferable to administer daily to once every 3 days. When using an expression vector, it can be administered about once a week.

[0043] The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example

[Example 1] Expression of HSP105 mRNA in a human cell line

HSP105 mRNA expression was examined using reverse transcription-PCR (RT-PCR). The human colon cancer cell line HCT116 was obtained from Dr. B. Vogelstein of Johns Hopkins University. Normal colon mucosa cDNA was purchased from Clontech.

[0045] RT-PCR was performed according to a known method. A human HSP105 gene PCR primer that amplifies a 407 bp fragment was designed and used to perform an RT-PCR reaction consisting of 33 amplification cycles at 94 ° C, 5 minutes of initial denaturation, and an annealing temperature of 58 ° C. It was. The HSP105 PCR primer sequence used was Sense: 5′-AGAGTAAAAGTCAAAGTG-3 ′ (SEQ ID NO: 5), Antisense: 5′-TTAGAAGGTCTTTCCCT-3 ′ (SEQ ID NO: 6). The j8-actin PCR primer sequences for the control experiments are sense: 5′-CCTCGCCTTTGCCGATCC-3 ′ (SEQ ID NO: 7), antisense: 5′-GGATCTTC ATGAGGTAGTC AGTC-3 ′ (SEQ ID NO: 8).

[0046] After normalization with β-actin mRNA as a control, the expression of HSP105 mRNA was compared in human cell lines. As a result, HSP105 mRNA was strongly expressed in the HCT116 cell line compared to normal colon mucosa (Fig. 1).

[Example 2] Inhibition of HSP105 protein expression and induction of apoptosis using small interference RNA

A small interference RNA (siRNA) consisting of 21 bases of mRNA (138-158) of heat shock protein 105 (HSP105) was designed and purchased from Dharmacon (Fig. 2). Also as a control Luciferase siRNA was also purchased from Dharmacon. All were dissolved in an annealing buffer (100 mM KOAc, 30 mM HEPES-KOH pH 7.4, 2 mM MgOAc) to 20 μ 20.

[0048] The HSP105 siRNA sequence is sense: 5'-UUGGCUGCAACUCCGAUUGdTdT-3 '(SEQ ID NO: 1), and antisense: 5'-CAAUCGGAGUUGCAGCCAAdTdT-3' (SEQ ID NO: 2).

The Luciferase siRNA sequence is sense: 5'-CGUACGCGGAAUACUUCGAdTdT-3 '(SEQ ID NO: 3), antisense: 5'-UCGAAGUAUUCCGCGUACGdTdT-3' (SEQ ID NO: 4).

[0049] Add 4 μ 1 of oligofectamine (invitrogen) to 16 μ 1 of opti-MEM (GIBCO) and let stand at room temperature for 5 minutes, then control this to opt MEM 180 μ 1 and opti-MEM 175 μ 1 Mix with a solution containing lOOpmol (5 μ 1) each of luciferase-siRNA and HSP105-siRNA for 20 minutes at room temperature. Apply this solution (200 μ1) to a 6-well plate the day before with 2 ml of 10% FCS DME M medium at 1 × 10 ⁵ / well, wash once with opti-MEM, and add 300 μ1 of opt-MEM. It was added to each well of human colon cancer cell lines HCT116 (strain with wild type p53) and HCT116 (wild type p53 deficient strain). After 4 hours, add 2 ml of 10% FCS DMEM medium.After 24 and 48 hours, collect cells by trypsin treatment.A part of the cells for flowcytometry and the rest for Western blotting are lysis buffer (20 mM Tris-HC1 pH7 .4, 10% glycerol, 1% NP-40 [Roche], 200 mM NaCl, ImM Na VO, Protease inhibitor cocktail tablet 1 tablet (Roche))

3 4

A cell lysate was prepared.

[0050] Western blotting was performed by a known method. The sample was electrophoresed on a 7% acrylamide gel, transferred to a nitrocellulose membrane (Bio Rad), and blocked with a 0.2% Tween20-TBS solution containing 5% skim milk and 1% BSA (4 ° C, overnight). HSP105 Usagi polyclonal antibody (Santa Cruz, California) and mouse monoclonal j8-actin antibody (Sigma) as a primary antibody were incubated at room temperature for 1 hour at room temperature. After washing, HRP-labeled anti-mouse antibody, HRP-labeled anti-rabbit The antibody (Amersham Bioscience) was incubated for 30 minutes. After washing, ECL Western Blottiong detection Reagents (Amersham Bioscience) was extracted.

[0051] Human colon cancer cell lines HCT116 (wild type p53-bearing strain) and HCT116 (wild type p53 deficient) The expression of HSP105 protein in the losing strain was equally decreased over time. In the Ludf erase siRNA treatment group used as a control, HSP105 expression did not decrease (Fig. 3).

Early apoptosis changes were examined with a flowcytometer using the ANNEXIN V-FITC detection kit (Bio Vision Inc.). Human colon cancer cell lines HCT116 (wild type p53-bearing strain) and HCT116 (wild type p53-deficient strain) were more effective in the wild-type p53-bearing strain in which apoptosis was induced (48 hours) % Of apoptosis: HCT116 p53- /-Oligofectamine only 12.0% Luciferase- siRNA treatment 8.4% HSP105- siRNA treatment 18.1% HCT116 p53 + / + Oligofectamine only 11.2% Luciferase- siRNA treatment 9.6% HSP105- siRNA treatment 51.1%) ( Figures 4 and 5).

[0052] [Example 3] Combination experiment of siRNA and anticancer agent

Furthermore, in Example 2, 12 hours after addition of siRNA, adriamycin was added to each well at a concentration of 200 ng / ml, and the effect was examined.

[0053]% of apoptosis after HSP105-siRNA treatment [36 hours after addition of adriamycin]% of apoptosis: HCT116 p53- /-27.8% HCT116 p53 + / + 78.4%) (Figure 6).

[Example 4] Apoptosis induction experiment in other human cell lines

In human colon cancer cell line SW620 and human liver cancer SK-Hepl, the effect was examined using siRNA as described in Example 2. In the SK-Hepl experiment, GFP siRNA was purchased from QIAGEN and used as a control siRNA. The GFP siRNA sequence is sense: 5′-GCAAGCUGAC CCUGAAGUUCAU-3 ′ (SEQ ID NO: 9), antisense: 5′-GAACUUCAGGGUCAGCU UGCCG-3 ′ (SEQ ID NO: 10).

[0055] The human colon cancer cell line SW620 showed the most decreased expression of HSP105 protein after 24 hours, and apoptosis was strongly induced at this time. This is probably due to the rapid growth of SW620, and after 48 hours, cells with strong enough siRNAs proliferated, so this result was relatively obtained. In human liver cancer SK-Hepl, the expression of HSP105 was suppressed most and apoptosis was strongly induced 48 hours later (Figs. 7 to 9).

[0056] In addition, apoptosis induction experiments were performed in the same manner as described above in human spleen cancer cell line PK8, gastric cancer cell lines 細胞 -ΠΙ, and ΜΚΝ28. Human spleen cancer cell line ΡΚ8, gastric cancer cell line ΚΑΤ In 0-111 and MKN28, the expression of HSP105 was suppressed 48 hours later, and apoptosis was induced (FIG. 10).

[Example 5] In vivo experiment

2 × 10 ⁶ human gastric cancer cell line KATO-III was dissolved in 100 μl of HBSS and injected subcutaneously into the back of NOD SCID mice. When the tumor became about 6 mm in size, a siRNA (HSP105 siRNA represented by SEQ ID NO: 1 and SEQ ID NO: 2 described in Example 2) solution was injected into the tumor every 3 days. It was synthesized with siRNA 1 nmol + 20 μ1 oligofectamin + glucose water to a final concentration of 5% grape solution 60 1. Following the volume of 4 tumors each with control siRNA.

V = LXW ² X 0.5

(V indicates volume, L indicates length, W indicates width)

Figure 11 shows the measurement results. As shown in Fig. 11, HSP105-siRNA clearly inhibited tumor growth.

[Example 6] Effect of siRNA in normal cells

We examined the effect of HSP105-siRNA on human normal cells (search for side effects). Using normal human fibrob last TURU and MORI (provided by Prof. Yamazumi of Kumamoto University), 100 pmol each of control GFP-siRNA and HSP105-si RNA in the same manner as described in Example 2 In addition, 3 days later, Western blotting and flowcytom etry (HSP105-siRNA group) using Annexin V were performed. The obtained results are shown in FIG. As can be seen from the results shown in FIG. 12, HSP105_siRNA suppressed the expression of HSP105 protein even in normal cells, but did not induce apoptosis. Furthermore, this protein expression suppression was also transient, and the strength recovered after about 10 days after the introduction of siRNA.

Industrial applicability

[0059] The antitumor agent of the present invention is expected to have a conventional antitumor effect by targeting a tumor-specific antigen using a new means. The effect of the antitumor agent of the present invention was enhanced by the combined use with other anticancer agents. In addition, in the gene therapy using the wild-type p53 expression vector as a state-of-the-art treatment, the therapeutic effect is low in malignant tumors expressing wild-type P53, but the antitumor agent of the present invention is Expresses wild-type p53 It has been demonstrated to be more effective in cancer cell lines. In addition, combined use with this gene therapy can be expected to have a synergistic effect on cancer with mutant p53. As described above, the antitumor agent of the present invention can be used as a new malignant tumor therapeutic agent in the field of medical therapeutic agents.

Brief Description of Drawings

FIG. 1 shows the results of examining the expression of HSP105 in normal human colon and human colon cancer cell line HCT116 by RT-PCR. The expression of HSP105 mRNA was clearly higher in HCT116 than in normal colon.

FIG. 2 shows the siRNA sequence of human HSP105.

FIG. 3 shows the result of confirming the expression of HSP105 protein after siRNA treatment by Western blotting. A: HCT116 p53-/-B: HCT116 p53 + / + (wild type) Both HCT116 wild type and HCT116 p53-/-decrease in HSP105 protein expression over time. On the other hand, HSP105 expression was not decreased by treatment with the control luciferase siRNA.

FIG. 4 shows detection of apoptosis 48 hours after siRNA treatment of HCT116 cell line by ANNEXIN V staining using flowcytometry. In both HCT116 wild type and HCT116 p53- /-, apoptosis was clearly induced by HSP1 05 siRNA treatment compared to treatment with Oligofectamine alone or luciferase siRNA, which was more prominent in HCT116 wild type.

[FIG. 5] FIG. 5 shows a graph of the results of FIG. 4 together with 24 hours later.

[FIG. 6] FIG. 6 shows detection of apoptosis 48 hours after siRNA treatment of HCT116 cell line by ANNEXIN V staining using flowcytometry (36 hours after addition of adriamycin 200 ng / ml). In combination with adriamycin, the cells could be more induced to apoptosis.

FIG. 7 shows the results of confirming protein expression of HSP105 by Western blotting after siRNA treatment of human colon cancer cell line SW620. SW620 showed the lowest HSP105 expression 24 hours after siRNA treatment.

FIG. 8 shows detection of apoptosis by ANNEXIN V staining of SW 620 using flowcytometry after siRNA treatment of human colon cancer cell line SW620. Apoptosis is treated with siRNA 2 [FIG. 9] FIG. 9 shows the protein expression of HSP 105 by Western blotting 48 hours after siRNA treatment of SK-Hepl and the detection of apoptosis by ANNEXIN V staining. SK_Hep 1 had a marked decrease in HSP105 protein expression 48 hours after siRNA treatment, and apoptosis was induced.

[FIG. 10] FIG. 10 shows protein expression of HSP105 by Western blotting 48 hours after siRNA treatment and apoptosis detection by ANNEXIN V staining in human spleen cancer cell line PK8, gastric cancer cell line M-ΠΙ, and MKN28 .

FIG. 11 shows the results of measuring the tumor volume over time after injecting HSP105 siRNA into the tumor on the back of the mouse. HSP105-siRNA suppressed tumor growth.

[Fig. 12] Fig. 12 shows the results of HSP105 protein expression by Western blotting after siRNA treatment of human normal fibroblast TURU and MORI, and flowcytometry after siRNA treatment of human normal fibroblast TURU and MORI. Shows detection of apoptosis by ANNEXIN V staining.

Claims

The scope of the claims

[1] An antitumor agent comprising a factor capable of suppressing the expression of HSP105 by RNAi.

[2] An agent for inducing apoptosis in cancer cells, comprising a factor capable of suppressing the expression of HSP105 by RNAi.

[3] The agent according to claim 1 or 2, wherein the factor capable of suppressing the expression of HSP105 by RNAi is siRNA or shRNA.

[4] The drug according to any one of claims 1 to 3, which is used in combination with an anticancer drug.

[5] The agent according to any one of claims 1 to 4, which is used for a cancer cell expressing wild-type p53.

[6] The siRNA force according to any one of claims 1 to 5, wherein the siRNA force is a double-stranded RNA having the same force as the RNA having the base sequence described in SEQ ID NO: 1 and the RNA having the base sequence described in SEQ ID NO: 2. Drugs.

7. The drug according to any one of claims 1 to 6, which is used for the treatment of colorectal cancer, knee cancer, esophageal cancer, breast cancer, or malignant melanoma.

[8] A double-stranded RNA comprising RNA having the base sequence set forth in SEQ ID NO: 1 and RNA having the base sequence set forth in SEQ ID NO: 2.