JPWO2006016574A1 - Antitumor agent using RNAi - Google Patents

Abstract

An object of the present invention is to provide a novel antitumor agent utilizing the RNAi phenomenon. ADVANTAGE OF THE INVENTION According to this invention, the antitumor agent containing the factor which can suppress the expression of HSP105 by RNAi is provided.

Description

  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.

  RNAi is a phenomenon in which a double-stranded RNA consisting of sense RNA and antisense RNA that is homologous to a specific region of a gene whose function is to be inhibited interferes with the homologous portion of the target gene transcript mRNA. It was first proposed by the experiment used. Later, in 2001, it was found that 21-23 base pair double-stranded RNA can induce RNAi effect in mammalian cells without showing cytotoxicity.

  Methods for suppressing the function of genes with known base sequences include homologous recombination gene knockout method, antisense method, ribozyme method, etc., but compared with such conventional gene function analysis methods, RNA The interference method is simple and inexpensive, and can analyze the function of a gene in a short time, and has the advantage of knowing at least 19 bases of the gene sequence to be inhibited. Furthermore, siRNA expression vectors have also 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 a gene product, and its application to medicine has been reported, but there are few reports that it was actually effective.

  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 considered that it binds to various proteins and is involved in the action of protecting against degradation and suppressing / activating the function. In addition, it has been shown that expression in mouse embryos is transiently increased during embryonic period (Hatayama. T. et al. Cell Struct. Funct. 22,517-525. (1997)). Overexpression of HSP105 in rat neurons has also been shown to suppress apoptosis induced by various stresses (Hatayama. T. et al. Biochem. Biophys. Res. Commun. 288, 528-534. 2001)). The present inventors have identified HSP105 in the SEREX method using serum of patients with pancreatic cancer and colorectal cancer, and this indicates that IgG antibodies against HSP105 are present 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 not only in pancreatic cancer and colon cancer but also in various human tumors (Kai. M. et al. Oncol. Rep. 10.1777-1782. (2003)). On the other hand, it was found that the expression was highest in the testis in normal human tissues, and weak expression was also observed in some organs such as the brain.

  In cancer, multidisciplinary treatment using various methods is performed as a difficult surgical case and postoperative treatment, but definitive treatment methods including side effects have not been established yet. Currently. 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.

  The present inventors considered that HSP105 has an oncogene-like function, and that the main one is to suppress apoptosis, since it was transformed by introducing the HSP105 gene into mouse NIH3T3 cells. 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 cancer cells expressing wild-type p53, and the effect was increased when used in combination with the anticancer drug adriamycin.

  Based on the above results, gene therapy using the introduction of wild-type p53 gene to cause apoptosis of cancer cells has already been performed, but cancer that expressed wild-type p53, which is considered to be ineffective for this gene therapy On the other hand, HSP105 siRNA is considered to be effective, and an additive / synergistic effect can be expected by combining this gene therapy with cancer having mutant p53.

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

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 agent of the present invention is used against cancer cells that express wild-type p53.

Preferably, the siRNA is a double-stranded RNA composed of 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.
Preferably, the agent of the present invention is used for the treatment of colorectal cancer, pancreatic cancer, esophageal cancer, breast cancer, malignant melanoma and the like that highly express HSP105.

  According to still 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.

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.

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 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 apoptosis inducer in cancer cells.

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 agent 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).

  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 in the present invention, can be used. Such siRNA can suppress gene expression by being expressed in a cell, and can suppress the expression of a target gene of the siRNA (HSP105 gene in the present invention).

  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 or biochemically synthesized, synthesized in an organism, or about 40 bases. This refers to a short double-stranded RNA of 10 base pairs or more formed by decomposing the above double-stranded RNA in the body, and usually has a 5′-phosphate, 3′-OH structure, and 3 ′ The end protrudes about 2 bases. A protein specific to the siRNA is bound to form RISC (RNA-induced-silencing-complex). This complex recognizes and binds to mRNA having the same sequence as siRNA, and cleaves mRNA at the center of siRNA by RNaseIII-like enzyme activity.

  It is preferable that the siRNA sequence and the mRNA sequence to be cleaved as a target coincide 100%. However, in the case where the bases at positions deviating from the center of the siRNA do not match, the cleavage activity by RNAi often remains partially, so it does not necessarily have to match 100%.

  The region having homology between the siRNA base sequence and the HSP105 gene base sequence whose expression is to be suppressed preferably does not include the translation start region of the HSP105 gene. This is because various transcription factors and translation factors are expected to bind to the translation initiation region, and therefore siRNA cannot effectively bind 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.

  In the present invention, siRNA can be used as a factor causing RNAi, and a factor that generates siRNA (for example, dsRNA having 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, still more preferably 90% or more, particularly preferably 95% with respect to a part of the nucleic acid sequence of the HSP105 gene. As described above, RNA containing a double-stranded portion or a variant thereof containing a sequence having 100% homology can be used most preferably. 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.

  Specific examples of siRNA that can be used in the present invention include RNA having a base sequence of 21 bases corresponding to positions 138 to 158 of HSP105 mRNA, specifically, the base sequence set forth in SEQ ID NO: 1. A double-stranded RNA consisting of RNA having an RNA and RNA having the base sequence set forth in SEQ ID NO: 2 is exemplified, but is not limited thereto. The base sequence of the HSP105 gene is known and is described in, for example, NCBI, Nucleotide Sequence Database accession No. AB003334.

  According to another aspect of the present invention, shRNA (short hairpin RNA) having a short hairpin structure having a protruding portion at the 3 ′ end can be used as a factor capable of suppressing the expression of HSP105 by RNAi. shRNA refers to a molecule of about 20 base pairs or more that has a double-stranded structure in a molecule and a hairpin-like structure by including a partially palindromic base sequence in a single-stranded RNA. To tell. Such shRNA, after being introduced into the cell, is degraded to a length of about 20 bases (typically, for example, 21 bases, 22 bases, 23 bases) in the cell, and causes RNAi similarly to siRNA. be able to. As described above, shRNA causes RNAi similarly to siRNA, and thus can be used effectively in the present invention.

  The shRNA preferably has a 3 ′ overhang. 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.

  As described above, in the present invention, siRNA or shRNA can be used as a factor that can suppress the expression of HSP105 by RNAi. The advantages of siRNA are as follows: (1) Since RNA itself is not integrated into the chromosome of normal cells even when introduced into cells, it is not a treatment that causes mutations transmitted to offspring, and is highly safe, and ( 2) Short double-stranded RNA is relatively easy to chemically synthesize and is more stable when double-stranded. Further, as an advantage of shRNA, when treatment is performed by suppressing gene expression for a long period of time, a vector that transcribes shRNA in a cell can be prepared and introduced into the cell.

  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, or the DNA sequence of the sense strand and antisense strand. A DNA having a hairpin structure ligated in the reverse direction can also be prepared by synthesizing RNA in vitro with T7 RNA polymerase. For synthesis in vitro, antisense and sense RNAs can be synthesized from template DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is induced and the expression of HSP105 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.).

  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 cell of the present invention may transiently or stably express a factor that causes RNAi. The expression vector and cell type described above are not particularly limited, but are preferably those that can be used for therapy.

  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, suppression of tumor metastasis, and the like. Clinically, cancer and / or tumor prevention and / or It is meant to encompass all of the treatments.

  The type of cancer for which the antitumor agent of the present invention can be used is not particularly limited, and includes all benign tumors and malignant tumors. Specific examples of cancer include malignant melanoma, malignant lymphoma, digestive organ cancer, lung cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, colon cancer, ureteral tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, breast cancer, liver Cancer, pancreatic 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, Kaposi's sarcoma, hemangioma, leukemia , 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.

  The agent of the present invention can be used in combination with other anticancer agents. In such cases, the agents of the present invention can be administered with certain anticancer agents or separately before and after. By administering another anticancer agent as described above, it is possible to effectively exert a therapeutic or prophylactic effect on cancer cells that are resistant to such an anticancer agent.

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 (for example, methotrexate, 6-mercaptopurine, fluorouracil (5-FU), etc.)
(3) DNA topoisomerase inhibitor (for example, camptothecin, etoposide, etc.)
(4) Tubulin agonists (vinblastine, vincristine, etc.)
(5) Platinum compounds (cisplatin, carboplatin, etc.)
(6) Anticancer antibiotics (adriamycin, daunorubicin, mitomycin C, bleomycin, etc.)
(7) Hormonal agents (tamoxifen, leuprorelin, etc.)
(8) Biologics (asparaginase, interferon, etc.)
(9) Immunostimulator (Lentinan derived from shiitake mushroom)

  The method of administering the agent of the present invention includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, local administration to the affected area, And administration directly to the affected area.

  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, buffers, delivery vehicles. , Diluents, carriers, excipients and / or pharmaceutical adjuvants and the like.

  Although the formulation form of the chemical | medical agent of this invention is not specifically limited, For example, a liquid agent, an injection, a sustained release agent etc. are mentioned. The solvent used for formulating the drug of the present invention as the above-mentioned preparation may be either aqueous or non-aqueous.

  Injections can be prepared by methods well known in the art. For example, after dissolving in an appropriate solvent (physiological saline, buffer solution such as PBS, sterilized water, etc.), sterilized by filtration with a filter, and then filled into a sterile container (eg, ampoule) 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.

  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, and include, for example, separate experimental medicine “Basic technology of gene therapy” Yodosha, 1996; separate experiment medicine “Gene transfer & expression analysis experimental method” Yodosha, 1997, etc. It is described in.

  In the case of a non-viral vector form, a method for introducing nucleic acid molecules using liposomes (liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.), microinjection method, gene gun (Gene Gun) ), A method of transferring a nucleic acid molecule into a cell together with a carrier (metal particle) can be used. Examples of expression vectors include pCAGGS, pBJ-CMV, pcDNA3.1, pZeoSV (available from Invitrogen or Stratagene).

  When lipofection is used, for example, Lipofectamine 2000, oligofectamine (silencer siRNA Transfection Kit, Gene Silencer siRNA Transfection Reagent) and the like can be used.

  The HVJ-liposome method involves encapsulating a nucleic acid molecule in a liposome made of a lipid bilayer and fusing the liposome with an inactivated Sendai virus (Hemagglutinating virus of Japan, HVJ). The HVJ-liposome preparation method is described in, 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 experimental method” Yodosha, 1997.

  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. Suppresses expression of HSP105 by RNAi in detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, Sendai virus, SV40 A gene can be introduced into a cell or tissue by introducing a DNA that expresses a possible factor and infecting the cell or tissue with the recombinant virus.

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

  The dosage of the drug of the present invention includes the purpose of use, the severity of the disease, the age, weight, sex, medical history of the patient, or the type of factor that can suppress the expression of HSP105 by RNAi as an active ingredient. In view of this, it can be determined by one skilled in the art.

  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 mg / kg, preferably about 1 ng to about 10 mg. When administered as a viral vector or a non-viral vector, the dose is usually 0.0001-100 mg, preferably 0.001-10 mg, more preferably 0.01-1 mg per adult.

  The administration frequency of the drug of the present invention can be administered, 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 to 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.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

[Example 1] Expression of HSP105 mRNA in a human cell line HSP105 mRNA expression using reverse transcription-PCR (RT-PCR) was examined. 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.

  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. The HSP105 PCR primer sequences used were sense: 5′-AGAGTAAAAGTCAAAGTG-3 ′ (SEQ ID NO: 5) and antisense: 5′-TTAAGAAGGTCTTTCCCT-3 ′ (SEQ ID NO: 6). The β-actin PCR primer sequence for the control experiment is sense: 5′-CCTCGCCTTTGCCGATCC-3 ′ (SEQ ID NO: 7), antisense: 5′-GGATCTTCATGAGGTAGTCAGTC-3 ′ (SEQ ID NO: 8).

  After normalization with the control β-actin mRNA, 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 heat shock protein 105 (HSP105) mRNA (138-158) was designed and purchased from Dharmacon (FIG. 2). Luciferase siRNA for control 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) so as to be 20 μM.

The HSP105 siRNA sequence is sense: 5′-UUGGCUGCAACUCCGAUUGdTdT-3 ′ (SEQ ID NO: 1), antisense: 5′-CAAUCGGAGUUGCAGCCAAdTdT-3 ′ (SEQ ID NO: 2).
The Luciferase siRNA sequence is sense: 5′-CGUACGCGGAAUACUUCGAdTdT-3 ′ (SEQ ID NO: 3), and antisense: 5′-UCGAAGUAUUCCGCGUACGdTdT-3 ′ (SEQ ID NO: 4).

Add 4 μl of oligofectamine (invitrogen) to 16 μl of opti-MEM (GIBCO), and let stand at room temperature for 5 minutes. 5μl) Mix with the added solution and leave at room temperature for 20 minutes. This solution (200 μl) was spread on a 6-well plate the day before with 2 ml of 10% FCS DMEM medium at 1 × 10 ⁵ / well, washed once with opti-MEM, and 300 μl of opti-MEM was added. It added to each well of cancer cell line HCT116 (wild type p53 possession strain) and HCT116 (wild type p53 deficiency strain). After 4 hours, 2 ml of 10% FCS DMEM medium was further added, and after 24 and 48 hours, cells were collected by trypsin treatment.A part was used for flowcytometry, and the rest was used for Western blotting in lysis buffer (20 mM Tris-HCl pH 7. 4, 10% glycerol, 1% NP-40 [Roche], 200 mM NaCl, 1 mM Na ₃ VO ₄ , and 1 protease inhibitor cocktail tablet [Roche]) were prepared to prepare cell lysate.

  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 (4 ° C, overnight) with a 0.2% Tween20-TBS solution containing 5% skim milk and 1% BSA. . HSP105 rabbit polyclonal antibody (Santa Cruz, California) and mouse monoclonal β-actin antibody (Sigma) as a primary antibody were incubated at room temperature for 1 hour at room temperature, washed, and then washed with HRP-labeled anti-mouse antibody, HRP-labeled anti-rabbit antibody ( Amersham Bioscience) was incubated for 30 minutes, washed, and then detected with ECL Western Blottiong detection Reagents (Amersham Bioscience).

The expression of HSP105 protein in human colorectal cancer cell lines HCT116 (carrying wild type p53) and HCT116 (wild type p53 deficient) was equally decreased over time. In the Luciferase siRNA treatment group used as a control, the expression of HSP105 did not decrease (FIG. 3).
Early apoptosis changes were examined with a flowcytometer using the ANNEXIN V-FITC detection kit (Bio Vision Inc.). Apoptosis was induced in both human colorectal cancer cell lines HCT116 (wild type p53-bearing strain) and HCT116 (wild type p53-deficient strain), but the wild type p53-bearing strain was more effective (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%) 4 and FIG. 5).

[Example 3] Combination experiment of siRNA and anticancer agent In Example 2, adriamycin was further added to each well at a concentration of 200 ng / ml 12 hours after addition of siRNA, and the effect was examined.

  The addition of adriamycin was able to induce apoptosis more effectively (% of apoptosis 48 hours after HSP105-siRNA treatment [36 hours after addition of adriamycin]: HCT116 p53-/-27.8% HCT116 p53 + / + 78.4%) ( FIG. 6).

[Example 4] Apoptosis induction experiment using other human cell lines The effects of human colon cancer cell line SW620 and human liver cancer SK-Hep1 were examined using siRNA as described in Example 2. In the SK-Hep1 experiment, GFP siRNA was purchased from QIAGEN as a control siRNA and used. The GFP siRNA sequence is sense: 5′-GCAAGCUGACCCUGAAGUUCAU-3 ′ (SEQ ID NO: 9), antisense: 5′-GAACUUCAGGGUCAGCUUGCCG-3 ′ (SEQ ID NO: 10).

  Human colorectal cancer cell line SW620 showed the lowest expression of HSP105 protein after 24 hours, and apoptosis was strongly induced at this time. This is probably because the growth of SW620 was fast, and after 48 hours, cells in which siRNA did not work sufficiently proliferated, so this result was relatively obtained. In human liver cancer SK-Hep1, the expression of HSP105 was most suppressed 48 hours later, and apoptosis was strongly induced (FIGS. 7 to 9).

  In addition, apoptosis induction experiments were performed in the same manner as described above in human pancreatic cancer cell line PK8, gastric cancer cell lines KATO-III, and MKN28. In human pancreatic cancer cell line PK8, gastric cancer cell lines KATO-III, 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. Synthesized with siRNA 1 nmol +20 μl oligofectamin + glucose water to a final concentration of 5% grape solution 60 μl. Volumes of 4 tumors each with control siRNA were followed.
V = L × W ² × 0.5
(V indicates volume, L indicates length, W indicates width)
The measurement results are shown in FIG. As can be seen from the results in FIG. 11, HSP105-siRNA clearly suppressed tumor growth.

[Example 6] Effect of siRNA in normal cells The effect of HSP105-siRNA in human normal cells (search for side effects) was observed. Using normal human fibroblast TURU and MORI (provided by Professor Yamazumi of Kumamoto University), add 100 pmol each of control GFP-siRNA and HSP105-siRNA by the same method as described in Example 2, Three days later, Western blotting and flowcytometry (HSP105-siRNA group) using Annexin V were performed. The obtained result is shown in FIG. As can be seen from the results 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 recovered about 10 days after the introduction of siRNA.

  The antitumor agent of the present invention is expected to have an antitumor effect that has never been achieved 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 malignant tumor expressing wild type p53 currently has a low therapeutic effect, but the antitumor agent of the present invention is a wild type. It has been demonstrated to be more effective in cancer cell lines expressing p53. In addition, an additive / synergistic effect can be expected for cancer having mutant p53 by using this gene therapy together. 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.

FIG. 1 shows the results of examining the expression of HSP105 in human normal 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 results of confirming the expression of HSP105 protein after siRNA treatment by Western blotting. A: HCT116 p53-/-B: HCT116 p53 + / + (wild type) In both HCT116 wild type and HCT116 p53-/-, the expression of HSP105 protein decreases equally over time. On the other hand, HSP105 expression was not decreased by treatment with luciferase siRNA as a control. FIG. 4 shows the 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 HSP105 siRNA treatment compared to treatment with Oligofectamine alone or luciferase siRNA, which was more prominent in HCT116 wild type. FIG. 5 shows a graph of the results of FIG. 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, cells could be induced to apoptosis more. FIG. 7 shows the results of confirming the 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 SW620 using flowcytometry after siRNA treatment of human colon cancer cell line SW620. Apoptosis was most induced 24 hours after siRNA treatment, and a decrease in protein expression correlated with the amount of apoptosis. FIG. 9 shows protein expression of HSP105 by western blotting 48 hours after siRNA treatment of SK-Hep1 and detection of apoptosis by ANNEXIN V staining. SK-Hep1 showed a marked decrease in HSP105 protein expression 48 hours after siRNA treatment, and apoptosis was induced. FIG. 10 shows the detection of HSP105 protein expression by Western blotting 48 hours after siRNA treatment and apoptosis by ANNEXIN V staining in human pancreatic cancer cell line PK8, gastric cancer cell line KATO-III, 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 shows HSP105 protein expression by Western blotting after siRNA treatment of human normal fibroblast TURU and MORI, and apoptosis by ANNEXIN V staining using flowcytometry after siRNA treatment of human normal fibroblast TURU and MORI. Indicates detection.

Claims (8)

An antitumor agent comprising a factor capable of suppressing the expression of HSP105 by RNAi. An apoptosis inducer in cancer cells, comprising a factor capable of suppressing the expression of HSP105 by RNAi. The agent according to claim 1 or 2, wherein the factor capable of suppressing the expression of HSP105 by RNAi is siRNA or shRNA. The drug according to any one of claims 1 to 3, which is used in combination with an anticancer drug. The agent according to any one of claims 1 to 4, which is used for cancer cells expressing wild-type p53. The drug according to any one of claims 1 to 5, wherein the siRNA is a double-stranded RNA composed of 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. The drug according to any one of claims 1 to 6, which is used for the treatment of colorectal cancer, pancreatic cancer, esophageal cancer, breast cancer, or malignant melanoma. A double-stranded RNA comprising 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.