KR101087614B1 - Pharmaceuticla composition comprising siRNA specific for reduced expression-1 for treating the BCNU-resistance glioblastoma multiforme - Google Patents

Abstract

The invention BCNU (1,3-Bis (2- chloroethyl) -1-nitrosourea, Carmustine) - resistance polymorphism glioblastoma therapy relates to a pharmaceutical composition, in particular stem cell marker of Rex-1 (r educed ex pression It relates to a pharmaceutical composition that induces apoptosis and tumor growth inhibition of BCNU-resistant polymorphous glioblastoma by including siRNA specific to Rex-1 as an active ingredient that inhibits the expression of -1). The pharmaceutical composition of the present invention can be usefully used for the treatment of BCNU-resistant polymorphic glioblastoma by co-administration with the BCNU.

BCNU-resistant polymorphic glioblastoma, Rex-1, siRNA

Description

Pharmaceuticla composition comprising siRNA specific for reduced expression-1 for treating the BCNU-resistance glioblastoma multiforme}

The present invention relates to a pharmaceutical composition for treating BCNU (1,3-Bis (2-chloroethyl) -1-nitrosourea, Carmustine) -resistant polymorphic glioblastoma.

In recent studies on cancer, a small group of cells with persistent cancer formation and growth capacity was reported in cancer cells. These cells are called tumor stem cells (CSCs). The tumor stem cells have similar capabilities to normal stem cells such as self-renewal and multi-potency, and express various transporters responsible for drug efflux. have. Recent studies have reported that genetic stem cell markers such as Oct4, Nango and Rex-1 ( zfp42) are expressed in various types of cancer cells such as breast cancer, ovarian cancer, lung cancer, and kidney cancer. Therefore, applying the basic concept of stem cell biology to target tumor stem cells is considered to improve the treatment of metastatic cancer. However , although Oct4, Nango, and Rex-1 ( zfp42) can be used as markers for tumor stem cells, it is still unclear on the tumor stem cell action of these genes.

Among the genetic stem cell markers identified so far Rex-1 (r educed ex pression -1) is a transcription factor zfp-42 (zinc finger protein- 42) F9 embryonic carcinoma is a kind of a family (embryonal carcinoma: EC) on the cell Was first discovered. Rex-1 gene is known as a marker of the degree of multipotency in human embryonic stem cells and mouse embryonic stem cells. In addition, Rex-1 targeted deletion of the gene (targeted deletion) during differentiation endoderm of the F9 EC cells (endodermal differentiation) is reduced, and gene silencing (gene silencing) by RNA interference (interference) of the Rex-1 gene is embryonic stem It has been reported to cause loss of self-renewal ability of the cells. Therefore, Rex-1 gene expression is expected to play an important role in regulating differentiation as well as pluripotency of embryonic stem cells. In addition, Rex-1 has been used as a multipotent marker of various stem cells, such as adult stem cells and amniotic fluid-derived cells having multipotency. In addition, recent reports suggest that Rex-1 may be used as a renal cell carcinoma stem cell marker by being expressed in human renal cell carcinoma. Therefore, from the point of view of tumor stem cells, the target therapy of Rex-1 gene may be useful for the treatment of tumors.

Human glioblastoma multiforme (GBM) has fatal consequences within a few weeks unless treated with aggressive variants compared to other brain tumors. Therefore, radiation therapy and chemotherapy are performed in addition to surgical procedures. BCNU (1,3-Bis (2-chloroethyl) -1-nitrosourea, Carmustine) is a chemical drug for glioblastoma multiforme that crosses the blood-brain barrier easily and cross-links DNA strands. It is most commonly used as an anticancer agent of human glioblastoma multiforme because it kills tumor cells through autokilling by inducing carbamoylation and alkylation. However, glioblastoma multiforme is known to be resistant to BCNU chemotherapy, and BCNU chemotherapy does not actually prolong median survival duaration in patients with glioblastoma multiforme. In addition, clinical studies of glioblastoma multiforme showed that BCNU chemotherapy increased survival when compared with radiotherapy alone. However, glioblastoma multiforme contains glioblastoma cells that are resistant to BCNU chemotherapy, resulting in multidrug resistance genes, DNA repair activity, and glutathione S-transferase. modifications such as transferase) and intracellular glutathione content are resistant to BCNU chemotherapy and result in persistent tumor recurrence. Recent studies have reported tumor recurrence by tumor stem cells present in glioblastoma multiforme, and these tumor stem cells also exhibit drug resistance. Recently, temozolomide (TMZ) has increased mismatch repair by O6) -alkylguanine (BCR) during BCNU-resistance, which has been shown in patients with BCNU-resistant brain tumors. As a new approach, TMZ chemotherapy is being proposed. Although TMZ has significantly increased patient survival for more than two years, long-term survival is still inadequate, and glioblastoma multiforme is known to contain tumor stem cells associated with overexpression of multidrug resistance (MDR) genes. Therefore, the development of new approaches targeting tumor stem cells would be considered a practical treatment for glioblastoma multiforme. Therefore, there is a need for a new approach to tumor therapy that focuses on the targeted treatment of drug-resistant tumor stem cell populations. Recently, the present inventors have reported that inhibition of Cl ^- channel (chloride channel) effectively promotes apoptosis and sensitivity to BCNU chemotherapy of glioblastoma multiforme glioblastoma (Kang MK & Kang SK, Biochem Biophys Res) Commun. 373 (4): 539-544, 2008).

Therefore, in the BCNU-resistant glioblastoma, which has been found to express a lot of mRNA and protein of Rex-1, the present inventors have found that siRNA (small) for the Rex-1 gene in vitro and in vivo is suppressed to suppress Rex-1 gene expression. The present invention is confirmed by inducing apoptosis and tumor growth inhibition by reducing the expression level of Rex-1 gene in polymorphogenic glioblastoma tumor stem cells by attempting combined-therapy using interfering RNA) and BCNU. Completed.

An object of the present invention is to provide a pharmaceutical composition for treating BCNU (1,3-Bis (2-chloroethyl) -1-nitrosourea, Carmustine) -resistant glioblastoma multiforme comprising Rex-1 specific siRNA as an active ingredient. .

Another object of the present invention is to provide a method for treating BCNU-resistant glioblastoma multiforme using Rex-1 specific siRNA.

In order to achieve the above object, the present invention is a BCNU (1,3-Bis (2-chloroethyl) -1-nitrosourea, Carmustine) -resistant glioblastoma multiform comprising a siRNA specific to the target sequence of Rex-1 as an active ingredient Provided is a therapeutic pharmaceutical composition.

The present invention also provides a pharmaceutical composition for treating BCNU-resistant polymorphic glioblastoma, comprising siRNA and BCNU specific to the target sequence of Rex-1.

The present invention also provides a method for treating BCNU-resistant glioblastoma multiforme comprising administering siRNA specific to the target sequence of Rex-1 to BCNU-resistant glioblastoma multiforme.

The present invention also provides a method for treating BCNU-resistant glioblastoma multiforme, comprising co-administering siRNA and BCNU specific to the target sequence of Rex-1 to BCNU-resistant glioblastoma multiforme.

The present invention also provides a method for treating BCNU-resistant glioblastoma multiforme, comprising co-administering siRNA, BCNU and Erk1 / 2 inhibitors specific for the target sequence of Rex-1 to BCNU-resistant glioblastoma multiforme.

The terms used in the present invention are explained.

As used herein, the term “siRNA” refers to a double-chain RNA capable of inducing RNA interference (RNAi) through cleavage of mRNA of a target gene, and refers to a sequence homologous to mRNA of a target gene. The branch consists of a sense RNA strand and an antisense RNA strand having a sequence complementary thereto. Since siRNA can inhibit expression of a target gene, it is provided by an efficient gene knockdown method or gene therapy method.

The phrase “inhibition of gene expression” refers to the ability of gene silencing for the Rex-1 gene of the siRNA of the invention. Inhibition of expression of the Rex-1 gene is achieved when the test value based on the control is about 90%, preferably 50%, more preferably 25-0%. Suitable assays include, for example, techniques known to those of skill in the art, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those skilled in the art. Investigation of used protein or mRNA levels.

In the present invention, the term "specific" or "specific" means the ability to inhibit only the target gene without affecting other genes in the cell, and is Rex-1 specific in the present invention.

Hereinafter, the present invention will be described in detail.

As also confirmed in the embodiment of the present invention, when 33 ㎍ / ㎖ BCNU treatment of the glioblastoma cells U87MG and A172, the cell viability was significantly reduced, but a small amount of cells were able to observe the survival . When treated with 33 μg / ml of BCNU for 3 days, U87MG and A172 cells showed low survival rates of 22.45% and 29.03%, respectively (see FIG. 1A), and also in these viable glioblastoma cells, ABCG2, TAP1 and SLCA42A Higher gene expression associated with the same drug transporter could be observed (see FIG. 1B). The results indicate that nestin-positive subpopulations of U87MG and A172 cell lines are resistant to BCNU and survive after BCNU chemotherapy.

The surviving U87MG and A172 cells showed a high immune response to nestin, a neural stem cell (NSC) marker, whereas a low immune response to GFAP, an astrocyte marker (FIG. 1c). In addition, these BCNU-resistant U87MG and A172 cells showed increased expression of GFAP while in vitro differentiation and decreased expression of Nestin (see FIG. 1D). Thus, BCNU-resistant Glioblastoma cells were found to be glial cells. It is shown to differentiate into cells having a phenotype of).

In addition, BCNU-resistant cells derived from U87MG and A172 showed significantly higher migration efficiencies compared to control cells (see FIG. 1E). These results supported previous results that BCNU-resistant cells derived from U87MG and A172 cells were tumor stem cells with pluripotency and high migration efficiency. In the case of tumor stem cells, BCNU-resistant cells exhibiting high migration efficiency are tumor stem cells.

In addition, to further investigate the ability of BCNU-resistant glioblastoma as a tumor stem cell in a specific embodiment of the present invention, human embryonic stem cells and adult stem cells, including Rex-1 , Nanog , Sox2 and Oct4 Stemness genes specific expression patterns were investigated. It was observed that mRNA and protein expression of Rex1, Nango, Sox2 and Oct4 were increased in BCNU-resistant glioblastoma cells compared to the control group (see FIGS. 2A and 2B). In addition, even in control glioblastoma cells, although the constitutively expressed stem cells and proteins, the expression level was increased in BCNU-resistant glioblastoma blasts. In particular, expression of the Rex-1 gene was significantly increased in U87MG and A172 derived BCNU-resistant cells. These results supported previous reports that BCNU-resistant cell populations derived from glioblastoma multiforme have tumor stem cell characteristics.

In order to investigate the correlation between Rex-1 and anticancer drug resistance in glioblastoma tumor stem cells, Rex-1 siRNA (silencing RNA) was treated before treatment with BCNU and cell viability was investigated. The cell viability of glioblastoma polymorphism of the treated group transfected with Rex-1 siRNA was significantly reduced (see FIG. 3A), and Rex-1 expression and cell survival were inhibited by Rex-1 siRNA / BCNU complex treatment (FIG. 3B and See FIG. 3C). In other words, RNA interference caused Rex-1 gene silencing to lose the self-replicating capacity of embryonic stem cells. Rex-1 siRNA / BCNU combination therapy in U87MG and A172 cells, a human glioblastoma cell line, was a BCNU - resistant tumor stem cell. It effectively inhibited the survival of in vitro .

In addition, in a specific embodiment of the present invention, Bax, Bcl-2, cytochrome C related to self-killing by Rex-1 siRNA / BCNU complex treatment in order to understand the pattern of growth inhibition by Rex-1 siRNA in glioblastoma multiforme The expression levels of P53, P21 and c-myc proteins decreased Bcl-2 protein expression, but Bax and cytochrome C protein expression and p53 and p21 protein expression associated with c-myc protein were affected by Rex-1 gene silencing. By inducing self-killing (see FIG. 4A). In addition, caspase-3 activity was markedly increased 1.8- and 1.9-fold in U87MG and A172 cells, respectively, by Rex-1 siRNA / BCNU combination therapy (see Figure 4b). The results are predicted that direct inhibition of Rex-1 gene by siRNA results in increased sensitivity to BCNU chemotherapy and promotes autophagy of glioblastoma multiforme.

In addition, MAPKs (Mitogen-Activated Protein Kinases) activity during self-killing is known to occur differently, such as self-killing promoting action by JNK (Jun kinase) and p38 MAPK and inhibiting self-killing by Erk. In a specific embodiment of the present invention, Western blots were performed to understand the mechanism of self-killing induced by Rex-1 siRNA / BCNU combination therapy. As a result, it was observed that Erk1 / 2 phosphorylation activity was significantly reduced in two types of glioblastoma multiforme, whereas phosphorylation activity of JNK and p38 was different depending on the cells (see FIG. 4C). From the above results, it was found that apoptosis of glioblastoma multiforme induced by Rex-1 siRNA / BCNU combination therapy was related to Erk1 / 2 inactivation, although p38 and JNK were activated differently in cell lines.

To investigate Rex-1 siRNA ability on cell growth inhibition and Erk1 / 2 inhibition in glioblastoma multiforme, Erk1 / 2 specific inhibitor PD98059 and Erk1 / 2 global inhibitor AG1478 on U87MG and A172-derived BCNU-resistant tumor stem cells Alternatively, Rex-1 siRNA was treated, and cell survival and Rex-1 expression patterns were examined. As a result, PD98059 and AG1478 reduced the cell survival of the glioblastoma cell population (see FIG. 5A). In this case, AG1478 was more effective in the treatment of BCNU-resistant glioblastoma tumor stem cells than PD98059. In particular, it was observed that apoptosis of most glioblastoma tumor cell groups was induced 3 days after Rex-1 siRNA transfection (see FIG. 5A). In addition, apoptosis by inhibitors and siRNA was proportional to the decrease in Erk1 / 2 protein and Rex-1 gene expression (see FIG. 5B). In conclusion, the highly expressed Rex-1 gene in BCNU-resistant glioblastoma tumor stem cells was effectively reduced by Rex-1 siRNA, resulting in decreased cell survival due to decreased Ekr1 / 2 activity. In other words, it was predicted that the Rex-1 gene induces Erk1 / 2 activity to maintain cell proliferative capacity of BCNU-resistant tumor stem cells present in glioblastomas U87MG and A172.

In a specific embodiment of the present invention, as a result of examining whether the in vitro result of growth inhibition by the Rex-1 gene target may show in vivo changes, tumors in which the Rex-1 siRNA was not injected were continuously grown. In contrast, growth of tumors injected with Rex-1 siRNA was observed to occur slowly (see FIG. 6A). In addition, it was observed that survival of mice injected with Rex-1 siRNA into tumors was prolonged (see FIG. 6B).

In addition, histopathological observations and downstream signaling systems were examined to determine the role of Rex-1 siRNA in in vivo xenograft tumor growth. Histopathologically, mouse-derived tumor tissues injected with intratumoral Rex-1 siRNA were able to observe significant cell destruction (see FIG. 6C). In addition, the expression of proteins related to self-killing such as Bax, cytochrome C, segmental caspase-3 and PARP was also significantly increased in tumor tissues of mice injected with Rex-1 siRNA, leading to self-killing (FIG. 6d). In addition, expression of stem genes in tumors injected with Rex-1 siRNA was reduced (see FIG. 6E). GFAP was also induced and Rex-1 and Nestin proteins were significantly reduced (see FIG. 6F). From these results, it is predicted that the reduction of Rex-1 gene expression inhibits the growth of glioblastoma by inducing cell death and inducing apoptosis of tumor stem cells expressing Nestin and stem cells. In conclusion, it was confirmed that Rex-1 siRNA significantly delayed tumor growth by effectively reducing tumor stem cells in vivo .

Thus, the Rex-1 siRNA of the present invention can be effectively used for the treatment of BCNU-resistant polymorphic glioblastoma.

 The present invention provides a pharmaceutical composition for treating BCNU (1,3-Bis (2-chloroethyl) -1-nitrosourea, Carmustine) -resistant glioblastoma multiforme comprising siRNA specific to the target sequence of Rex-1 as an active ingredient. do.

The target sequence of Rex-1 is characterized in that SEQ ID NO: 17 or SEQ ID NO: 18. The siRNA may comprise a single RNA strand of stem-loop structure comprising an independent sense RNA strand and a complementary antisense RNA strand homologous to the target sequence, or wherein the sense RNA strand and antisense RNA strand are connected by a loop. The sense RNA strand is preferably SEQ ID NO: 17 or SEQ ID NO: 18, but is not limited thereto.

The siRNA is not limited to completely paired double-chain RNA portion paired with RNA, and may have a hairpin structure that forms a stem-loop structure, in particular, shRNA (short hairpin RNA). Refer. On the other hand, the double chain or stem region may include a portion not paired by mismatch (corresponding base is not complementary), bulge (the base does not correspond to one chain) and the like. The total length is 10 to 80 bases, preferably 15 to 60 bases, more preferably 20 to 40 bases. In addition, the loop region has no special meaning in the sequence, and only has 3 to 10 bases in order to connect the sense sequence and the antisense sequence at appropriate intervals. Examples that have been widely used as loop regions of siRNAs are as follows: AUG (Sui et al., Proc. Natl. Acad. Sci . USA 99 (8): 5515- 5520, 2002), CCC, CCACC or CCACACC ( Paul et al., Nature Biotechnology 20: 505-508, 2002), UUCG (Lee et al., Nature Biotechnology 20: 500-505), CTCGAG, AAGCUU (Editors of Nature Cell Biology Whither RNAi, Nat Cell Biol. 5: 489-490, 2003), UUCAAGAGA (Yu et al., Proc. Natl. Acad. Sci. USA 99 (9): 6047-6052, 2002) and TTGATATCCG (default spacer at www.genscript.com). siRNA terminal structures can be either blunt or cohesive. The cohesive end structure can be both a 3 'protruding structure and a 5' end protruding structure, and the number of protruding bases is not limited. For example, the number of bases may be 1 to 8 bases, preferably 2 to 6 bases. In addition, siRNA is a low-molecular RNA (for example, natural RNA molecules such as tRNA, rRNA, viral RNA or artificial RNA molecules such as tRNA, rRNA, viral RNA, etc.) in a range capable of maintaining the expression inhibitory effect of the target gene. ) May be included. The siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a stem loop type structure in which one terminal portion of the double chain RNA is connected by a linker RNA. The length of the linker is not particularly limited as long as it does not interfere with pairing of stem portions.

The siRNA is usually administered as a pharmaceutical composition. The administration can be carried out by known methods in which the nucleic acid is introduced into the target cell in the desired in vitro or in vivo . At this time, the introduction of siRNA into the cell is not particularly limited, but may be directly synthesized and then directly incorporated into the host cell, or may be siRNA expression vector or PCR-based siRNA expression cassette prepared to express the siRNA in the cell. It is preferable to carry out by transforming or infecting the cells, and the expression vector is not particularly limited. The vector expressing siRNA can use any conventionally known siRNA expression vector capable of expressing the target sequence of the present invention as siRNA. For example, Promega products include the GeneClip ™ U1 Hairpin Cloning System (Cat. # 'S C8750, C8760, C8770, C8780, C8790), the siLentGene ™ U6 Cassette RNA Interference System (Cat. # C7800), and the T7 RiboMAX ™ Express RNAi System (Cat # P1700), siSTRIKE ™ U6 Hairpin Cloning Systems (Cat. # 'S C7890, C7900, C7910, C7920) and siLentGene ™ -2 U6 Hairpin Cloning Systems (Cat. #' S C7860, C8060, C8070, C8080) GenRNA products available include pRNA-U6.1 (Cat. # 'S SD1201, SD1202, SD1207), pRNATin-H1.2 (Cat. #' S SD1223, 1224) and pRNA-H1.1 / Adeno (Cat # SD12009).

Meanwhile, commonly used gene transduction techniques into target cells include calcium phosphate, DEAE-dextran, electroporation and microinjection and viral methods (Graham, FL and van der Eb, AJ (1973) Virol. 52, 456; McCutchan, JH and Pagano, JS (1968), J. Natl. Cancer Inst. 41, 35 1; Chu, G. et al. (1987), Nucl. Acids Res. 15, 1311; Fraley, R. et al. (1980) ), J. Biol. Chem. 255, 10431; Capecchi, MR (1980), Cell 22, 479). A recent addition to these techniques for introducing nucleotides into cells is the use of cationic liposomes (Felgner, P.L. et al. (1987), Proc. Nati. Acad. Sci. USA 84, 7413). Commercially available cationic lipid preparations are, for example, Tfx 50 (Promega) or Lipofectamin 20O (Life Technologies). The method of directly incorporating siRNA capable of hybrid binding to the Rex-1 gene directly into the cell is not particularly limited thereto, but may mix 2 to 6 µg of cationic liposomes per µg of siRNA, and lipofection for 15 to 40 minutes. (lipofection) is preferably performed.

The pharmaceutical composition may be administered in any suitable manner, such as by injection, oral, topical, nasal, rectal application, and the like. The carrier can be any suitable pharmaceutical carrier. Preferably, a carrier is used that can increase the efficiency with which the RNA molecule is introduced into the target-cell. Suitable examples of such carriers are liposomes, especially cationic liposomes. More preferred method of administration is injection.

The pharmaceutical composition may be used for therapeutic application as a human medicine or a veterinary medicine, and the pharmaceutical composition may be in the form of a liquid such as an injectable solution, cream, ointment, tablet, suspension, and the like.

The pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances which facilitate the administration of pharmaceutically active substances. For example, an excipient can impart a shape or viscosity or can also act as a diluent. Suitable excipients include, but are not limited to, stabilizers, humectants, and emulsifiers, salts capable of varying osmolality, encapsulating agents, buffers, skin penetration promoters. Excipients and formulations for oral and parenteral drug delivery are presented in Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing (2000).

The effective concentration of siRNA is an amount sufficient to provide a reduction in the expression of the Rex-1 gene compared to the normal expression level detected in the absence of the desired effect, for example siRNA. siRNA may be introduced in an amount that allows delivery of at least one copy per cell. The higher the amount of double stranded material introduced (e.g., at least 5, at least 10, at least 100, at least 500, or at least 1000 copies per cell), the higher the inhibitory efficiency, and the lower the amount of introduction for specific applications. It may be advantageous.

The present invention also provides a pharmaceutical composition for treating BCNU-resistant polymorphic glioblastoma, comprising siRNA and BCNU specific to the target sequence of Rex-1.

In a specific embodiment of the present invention, BCNU / Rex-1 siRNA complex treatment of BCNU - resistant polymorphic glioblastoma tumor stem cells inhibited Rex-1 expression and cell viability (see FIGS. 3B and 3C). In other words, RNA interference caused Rex-1 gene silencing to lose the self-replicating capacity of embryonic stem cells, and Rex-1 siRNA / BCNU combination treatment effectively inhibited the in vitro survival of BCNU - resistant tumor stem cells. Thus, the combination of siRNA and BCNU specific to the target sequence of Rex-1 of the present invention can be usefully used for the treatment of BCNU-resistant polymorphic glioblastoma.

In addition, the present invention provides a method for treating BCNU-resistant glioblastoma multiforme comprising administering siRNA specific to the target sequence of Rex-1 to BCNU-resistant glioblastoma multiforme.

In a specific embodiment of the present invention, the cell survival rate of the polymorphic glioblastoma of the treatment group in which Rex-1 siRNA was transfected with BCNU-resistant glioblastoma tumor stem cells was significantly reduced (see FIG. 3A). In other words, RNA interference caused Rex-1 gene silencing to lose the self-replicating capacity of embryonic stem cells and effectively inhibited in vitro survival of BCNU-resistant tumor stem cells. Therefore, siRNA specific for the target sequence of Rex-1 of the present invention can be usefully used for the treatment of BCNU-resistant polymorphic glioblastoma.

The target sequence of Rex-1 is characterized in that SEQ ID NO: 17 or SEQ ID NO: 18. The siRNA may comprise a single RNA strand of stem-loop structure comprising an independent sense RNA strand and a complementary antisense RNA strand homologous to the target sequence, or wherein the sense RNA strand and antisense RNA strand are connected by a loop. The sense RNA strand is preferably SEQ ID NO: 17 or SEQ ID NO: 18, but is not limited thereto.

The BCNU-resistant glioblastoma multiforme may be isolated cells or cultured cells expressing the Rex-1 gene, and may be included in mammals without being separated. Examples of mammals include non-human mammals (eg, dogs, cats, horses, pigs, sheep, cattle, goats, rodents; hamsters, mice, rats, and primates) and humans. In the present invention, U87MG and A172 were used as BCNU-resistant polymorphic glioblastomas expressing target genes.

In the present invention, "administration" means introducing a certain substance into a patient in any suitable way and the route of administration of the substance can be administered via any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. In addition, the pharmaceutical composition may be administered by any device that allows the active substance to migrate to the target cell.

In addition, the phrase "administration" means that the siRNA of the present invention is introduced by "systemic delivery" or "local delivery" to cells expressing the Rex-1 gene. "Whole body delivery" refers to delivery that causes widespread biodistribution of a compound in an organism. Some dosing techniques cause systemic delivery to certain compounds, others may not. Systemic delivery means that a useful, preferably therapeutically effective amount of a compound is exposed to most of the body. In general, in order to obtain a broad biodistribution, fast nonspecific cell binding or by fast disassembly or removal of the compound (e.g., the first passage organ (liver, lung, etc.) before reaching the disease site far from the site of administration. Life expectancy in the blood is required. Systemic delivery of nucleic acid-lipid particles may be any means known in the art, including intravenous, subcutaneous, intraperitoneal, and in preferred embodiments, systemic delivery of nucleic acid particles is directed to intravenous delivery. Is made by As used herein, "topical delivery" refers to the delivery of a compound directly to a target site in an organism. For example, the compound can be delivered locally by injection directly into a disease site, such as a tumor or other target site, such as an inflammatory site or target organ, such as the liver, heart, pancreas, kidney, or the like.

The present invention also provides a method for treating BCNU-resistant glioblastoma multiforme, comprising co-administering siRNA and BCNU specific to the target sequence of Rex-1 to BCNU-resistant glioblastoma multiforme.

The "co-administration" is a method of first performing siRNA treatment several hours before the administration of BCNU. This is because siRNA must be treated first to inhibit Rex-1 gene expression, thereby increasing BCNU sensitivity in glioblastoma multiforme.

In a specific embodiment of the present invention, BCNU / Rex-1 siRNA complex treatment of BCNU - resistant polymorphic glioblastoma tumor stem cells inhibited Rex-1 expression and cell viability (see FIGS. 3B and 3C). In other words, RNA interference caused Rex-1 gene silencing to lose the self-replicating capacity of embryonic stem cells, and Rex-1 siRNA / BCNU combination treatment effectively inhibited the in vitro survival of BCNU - resistant tumor stem cells. Thus, the combination of siRNA and BCNU specific to the target sequence of Rex-1 of the present invention can be usefully used for the treatment of BCNU-resistant polymorphic glioblastoma.

The present invention also provides a method for treating BCNU-resistant glioblastoma multiforme, comprising co-administering siRNA, BCNU and Erk1 / 2 inhibitors specific for the target sequence of Rex-1 to BCNU-resistant glioblastoma multiforme.

The Erk1 / 2 inhibitor is PD98059, an Erk1 / 2 specific inhibitor, or AG1478, an Erk1 / 2 wide area inhibitor.

In a specific embodiment of the present invention, BCNU-resistant tumor stem cells were treated with PD98059, an Erk1 / 2 specific inhibitor, and AG1478 or Rex-1 siRNA, an Erk1 / 2 global inhibitor, respectively, and cell survival and Rex-1 expression were examined. As a result, PD98059 and AG1478 reduced the cell survival of the glioblastoma cell population (see FIG. 5A). In particular, it was observed that apoptosis of most glioblastoma tumor cell groups was induced 3 days after Rex-1 siRNA transfection (see FIG. 5A). In addition, apoptosis by inhibitors and siRNA was proportional to the decrease in Erk1 / 2 protein and Rex-1 gene expression (see FIG. 5B). In conclusion, the highly expressed Rex-1 gene in BCNU-resistant glioblastoma tumor stem cells was effectively reduced by Rex-1 siRNA, resulting in decreased cell survival due to decreased Ekr1 / 2 activity. In other words, it was predicted that the Rex-1 gene induces Erk1 / 2 activity to maintain cell proliferation of BCNU-resistant tumor stem cells present in glioblastoma. Thus, the combination treatment of siRNA, BCNU and Erk1 / 2 inhibitors specific for the target sequence of Rex-1 of the present invention can be usefully used for the treatment of BCNU-resistant glioblastoma multiforme.

 The pharmaceutical composition of the present invention can be usefully used for the treatment of BCNU-resistant polymorphic glioblastoma by co-administration with the BCNU.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Characterization of BCNU-resistant Glioblastoma

<1-1> BCNU-resistant Survival Rate of BCNU-resistant Glioblastoma

Human glioblastoma cell lines U87MG and A172 were purchased from ATCC (USA) and used. The two cell lines were cultured under 37 ° C./5% CO ₂ conditions in DMEM medium (Gibco Laboratories, USA) containing 10% FBS and 100 units / ml penicillin-streptomycin. All experiments were carried out when the algebraic multiplier reached about 70% confluence. To investigate the drug susceptibility of the two cancer cell lines, U87MG and A172 cells were cultured in 24-well plates and BCNU (33 μg / ml; 1,3-bis (2-chloroethyl) -1-nitrosourea, Sigma Chemical Company, USA ) Was further incubated for 3 days at 37 ℃ / 5% CO ₂ conditions. The control group was treated with DMSO instead of BCNU. Cell viability was examined by trypan blue dye exclusion, and all experiments were repeated three times. All data were expressed as mean ± standard deviation (SD) through three replicates and statistically treated by two-tailed Student's t- test ( P <0.05).

As a result, when treated with 33 μg / ㎖ BCNU for 3 days as shown in Figure 1a, U87MG and A172 cells showed a low survival rate of 22.45% and 29.03%, respectively.

<1-2> BCNU-resistant Characteristics of BCNU-resistant Glioblastoma

Gene expression patterns associated with drug transporters such as ABCG2, TAP1 and SLCA42A in BCNU-resistant tumor cells and control BCNU-resistant tumor cells surviving in Example 1-1 were identified. Total RNA isolated using Trizol (Life Technologies, USA) was reverse transcribed into primary cDNA using oligo-dT primers and denatured at 95 ° C. for 5 minutes using 20 pM gene specific primer pairs in Table 1. Then, it amplified while repeating 35 times on the conditions of 95 degreeC 1 minute, an anneal temperature 1 minute, and 72 degreeC 1 minute. The reaction was further reacted at 72 ° C. for 7 minutes. The annealing temperature for each primer was performed at 54 ° C. for ABCG2, 60 ° C. for TAP1, 55 ° C. for SLC4AZ, and 55 ° C. for GAPDH. Each 6 μl of gene product was electrophoresed on 1% agarose gel and stained with EtBr and observed in UV light. At this time, GAPDH was used as an internal control.

As a result, as shown in FIG. 1B, the expression of genes related to drug transporters such as ABCG2, TAP1, and SLCA42A was high in surviving glioblastoma cells.

Gene name Forward primer Reverse primer SEQ ID NO: Sequence SEQ ID NO: Sequence ABCG2 SEQ ID NO: 1 5'-caggaggccttgggatactt-3 ' SEQ ID NO: 2 5'-gctatagaggcctggggatt-3 ' TAP1 SEQ ID NO: 3 5'-gggctgtaagcagtgggaacc-3 ' SEQ ID NO: 4 5'-caaggccctccaagtgtaaggg-3 ' SLC4AZ SEQ ID NO: 5 5'-tgactgccccagaaaagag-3 ' SEQ ID NO: 6 5'-atccccgataactatyggagag-3 ' GAPDH SEQ ID NO: 7 5'-catgaccacagtccatgccatcact-3 ' SEQ ID NO: 8 5'-tgaggtccaccaccctgttgctgta-3 '

<1-3> Characterization of Tumor Stem Cells of BCNU-resistant Glioblastoma

The BCNU-resistant tumor cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes in order to investigate the neural stem and glial marker protein expression patterns in BCNU-resistant tumor cells surviving in Example 1-1. . After washing with PBS, the cells were incubated at 4 ° C using anti-GFAP (1: 1500, DAKO Cytomation, Denmark) and anti-Nestin (1: 200, Sigma, USA) primary antibodies. The cells were washed again with PBS and incubated for 30 minutes with secondary antibodies of FITC, Texas-Red, or TRITC (1: 250, Molecular Probe, USA). Cell nuclei were stained with DAPI (4-6'diamidino-2-phenylindoline; Vector laboratories, UK) and examined by Confocal Microscopy (Leica Microsystems, USA). Immunostaining experiments were performed three times each.

As a result, as shown in Figure 1c, the surviving U87MG and A172 cells showed a high immune response to Nestin, a neural stem cell (NSC) marker, whereas GFAP, an astrocyte marker. Low immune response to

<1-4> Tumor Stem Cell Characterization of BCNU-resistant Glioblastoma

BCNU-resistant tumor cells surviving in Example 1-1 were disrupted with PRO-PREP ^™ Protein Extraction Solution (iNTRON Biotechnology, Inc., USA) and electrophoresed with the same amount (40 μg) of protein with 12% SDS-PAGE gel. Afterwards, they were transferred to nitrocellulose membranes (Sigma, USA) and immobilized with 5% skim milk (skim milk, Difco, USA) for 2 hours at room temperature. After inducing antigen-antibody reaction at 4 ° C using anti-GFAP (1: 1500, DAKO, USA) and anti-Nestin (1: 200, Sigma, USA) primary antibodies, HRP (horseradish peroxidase) was attached. Reacting with secondary antibodies (Cell Signaling Technology, USA) and detecting protein expression using an enhanced chemiluminescence solution kit (Amersham, UK), the Quantity-one 1-D analysis software (Bio-Rad, USA) The relative protein expression levels were examined.

As a result, as shown in FIG. 1D, the surviving BCNU-resistant U87MG and A172 cells increased the expression of GFAP by in vitro differentiation and decreased the expression of Nestin. Thus, BCNU-resistant glioblastoma multiforme cells It has been shown to differentiate into cells having a phenotype of glial cells.

<1-5> Cell migration assay of BCNU-resistant glioblastoma

BCNU-resistant tumor cells ( ⁵ × 10 ⁵ cells) surviving in Example 1-1 were injected into Costar transwell membranes (Corning, USA) and then transferred to 6-well plates and incubated overnight in a 37 ° C./CO ₂ incubator. Cells migrated to the lower chamber were stained with Harris hematoxylin solution (Sigma, USA) for 20 minutes, washed, and counted under an optical microscope (× 200).

As a result, as shown in Figure 1e, U87MG and A172-derived BCNU-resistant cells showed significantly higher migration efficiencies compared to control cells.

<Example 2> Rex-1 expression of BCNU-resistant glioblastoma

<2-1> Gene expression level investigation

To further investigate its ability as tumor stem cells in BCNU-resistant tumor cells surviving in Example 1-1, Rex-1 , Nanog , Sox2 and Oct4 , known as markers of human embryonic stem cells and adult stem cells, were prepared. Stemness genes specific expression patterns were investigated in a similar manner to Example 1-2 using the primer pairs in Table 2. Annealing temperature for each primer was 55 ℃ for Rex1, 53 ℃ for Oct4, 59 ℃ for Sox2, 59 ℃ for Nanog, 55 ℃ for GAPDH.

As a result, as shown in Figure 2a it was observed that the mRNA expression of Rex1, Nango, Sox2 and Oct4 is increased in BCNU-resistant glioblastoma cells compared with the control group. In particular, expression of the Rex-1 gene was significantly increased in U87MG and A172 derived BCNU-resistant cells.

Gene name Forward primer Reverse primer SEQ ID NO: Sequence SEQ ID NO: Sequence Rex1 SEQ ID NO: 9 5'-tgaaagcccacatcctaacg-3 ' SEQ ID NO: 10 5'-caagctatcctcctgctttgg-3 ' Oct4 SEQ ID NO: 11 5'-cgcacaactggcattgtcat-3 ' SEQ ID NO: 12 5'-ttctccttgatgtcacgcac-3 ' Sox2 SEQ ID NO: 13 5'-tacctcttcctcccactcca-3 ' SEQ ID NO: 14 5'-actctcctcttttgcacccc-3 ' NaNog SEQ ID NO: 15 5'-gctgagatgcctcacacggag-3 ' SEQ ID NO: 16 5'-tctgtttcttgactgggaccttgtc-3 ' GAPDH SEQ ID NO: 7 5'-catgaccacagtccatgccatcact-3 ' SEQ ID NO: 8 5'-tgaggtccaccaccctgttgctgta-3 '

<2-2> Protein expression level investigation

Specific expression patterns of stem genes in BCNU-resistant tumor cells surviving in Example 1-1 were shown in Rex1 (1: 1000, Abcam, UK), Nango (1: 1000, Abcam, UK), Sox2 (1: 1000, Chemicon, USA) and Oct4 (1: 1000, Santa Cruz Biotechnology, Germany) specific primary antibodies were used in the same manner as in Example 1-3.

As a result, as shown in Figure 2b it was observed that the protein expression of Rex1, Nango, Sox2 and Oct4 is increased in BCNU-resistant glioblastoma cells compared to the control. In particular, expression of the Rex-1 gene was significantly increased in U87MG and A172 derived BCNU-resistant cells.

These results supported previous reports that BCNU-resistant cell populations derived from glioblastoma multiforme have tumor stem cell characteristics.

Example 3 Effect of Target Therapy of Rex-1 Gene

<3-1> Transfection with Rex1 siRNA

To investigate the correlation between Rex-1 and anticancer drug resistance in BCNU-resistant glioblastoma tumor stem cells, transfection was performed with Rex-1 siRNA prior to BCNU treatment.

Specifically, siRNA transfection was performed using LipofectAMINE 2000 (Invitrogen, USA). Human glioblastoma U87MG and A172 cells were incubated in DMEM medium (complete medium) with 10% FBS and transferred to 6-well plates when reaching 60-70% confluence. After 24 hours of incubation, the medium was replaced with a medium free of FBS (serum-free medium), and 5 μg of siRNA (SEQ ID NO: 17: 5'-GAAGATGGGAAGCGCCAAG-3 '; SEQ ID NO: 18: 5'-CAGGGGGUUGUGGUUAAGCUCUU) was used with LipofectAMINE 2000. (Dharmacon Inc., USA) was transfected, replaced with complete medium after 5 hours and treated with 33 μg / ml of BCNU and incubated under 37 ° C./5% CO ₂ conditions for 48 hours. The negative control group was scrambled siRNA (SEQ ID NO: 19: 5'-TTCTCCGAACGTGTCACGT-3 ', Gima Biol Engineering Inc., China) The positive control group was treated with BCNU alone.

<3-2> Correlation between Rex-1 and Anticancer Resistance

<3-2-1> Cell survival rate investigation

The cell viability of the transfected cells of Example 3-1 was examined using a phase contrast microscope (Nikon, Japan).

As a result, as shown in Figure 3a, the cell survival rate of glioblastoma multiforme was significantly reduced by treatment with Rex-1 siRNA.

<3-2-2> Rex-1 expression level investigation

The mRNA expression level of Rex-1 of the transfected cells of Example 3-1 was examined by the method of Example 1-2 using primer pairs of SEQ ID NO: 9 and SEQ ID NO: 10.

As a result, as shown in FIG. 3B, the mRNA expression level of Rex-1 of the polymorphic glioblastoma transfected with Rex-1 siRNA was suppressed.

<3-3> Cell survival rate by treatment period

The transfected cells of Example 3-1 were recovered on the 1st, 2nd and 3rd day, and cell viability was examined by the method of Example 1-1.

As a result, as shown in Fig. 3c, the cell survival rate of Rex-1 siRNA-transfected glioblastoma multiforme was suppressed.

Example 4 Confirmation of Inhibition of Cell Growth and Survival Signals

<4-1> Confirmation of growth inhibition characteristics

Specific expression patterns of stem genes in BCNU-resistant tumor cells transfected in Example 3-1 were determined by Bax (1: 1000, Santa Cruz Biotechnology, Germany), Bcl-2 (1: 1000, Santa Cruz Biotechnology, Germany). ), Cytochrome C (1: 1000, Santa Cruz Biotechnology, Germany), P53 (1: 500, Santa Cruz Biotechnology, Germany), P21 (1: 500, Santa Cruz Biotechnology, Germany), c-myc (1: 1000, Santa Cruz Biotechnology, Germany) and GAPDH (1: 1000, Santa Cruz Biotechnology, Germany) internal control) specific primary antibodies were used in the same manner as in Example 1-3.

As a result, Bcl-2 protein expression was reduced as shown in Figure 4a, but Bax and cytochrome C protein expression and p53 and p21 protein associated with c-myc protein was increased by Rex-1 gene silencing.

<4-2> Caspase-3 activity measurement

The transfected cells of Example 3-1 were washed with HBSS (Hank's Balanced Salts, Modified) buffer, lysed with CytoBuster ^™ Protein Extraction Reagent (Novagen, USA) for 1 hour at 4 ° C and centrifuged for 5 minutes (12,000 rpm). ). Caspase-3 substrate (Chelbiochem, Germany) was added to 50 μl reaction buffer, incubated at 37 ° C. for 2 minutes, and color development was observed at 405 nm using a micro reader (Molecular Devices, USA).

As a result, as shown in Figure 4b by Rex-1 siRNA / BCNU combination therapy significantly increased 1.8 and 1.9 times in U87MG and A172 cells, respectively.

<4-3> Confirmation of features of self-killing mechanism

Expression of MAPKs (Mitogen-Activated Protein Kinases) related proteins in transfected BCNU-resistant tumor cells of Example 3-1 was determined by Erk1 / 2 (1: 1000, Cell Signaling Technology, USA), p38 (1: 1000, Examples using Cell Signaling Technology, USA), Stress-activated protein kinase / c-Jun NH2-terminal kinase: 1: 1000, Cell Signaling Technology, USA) and GAPDH (Internal Control) specific primary antibodies The investigation was carried out in a similar manner to 1-3.

As a result, as shown in FIG. 4c, Erk1 / 2 phosphorylation activity was significantly reduced in two types of glioblastoma multiforme, whereas phosphorylation activity of JNK and p38 was different depending on the cells.

<Example 5> Interference check of cell survival signal transmission system

<5-1> Cell survival rate investigation

Transfected BCNU-resistant tumor cells of Example 3-1 and 10 μM each of Erk1 / 2 specific inhibitor PD98059 (Sigma, USA) and Erk1 / 2 wide area inhibitor AG1478 (Chemicon, USA) instead of the transfection Then, 1, 2 and 3 days later, the cell viability was examined by the method of Example 1-1.

As a result, as shown in Fig. 5a, PDrk59, an Erk1 / 2 specific inhibitor, and AG1478, an Erk1 / 2 wide area inhibitor, reduced the cell survival of the glioblastoma cell population. In this case, AG1478 was more effective in the treatment of BCNU-resistant glioblastoma tumor stem cells than PD98059. In particular, it was observed that apoptosis of most glioblastoma cell populations was induced 3 days after Rex-1 siRNA transfection.

<5-2> Erk1 / 2 And Rex-1 expression rate investigation

The relationship between the apoptosis rate by the inhibitor and siRNA and the expression level of Erk1 / 2 protein and Rex-1 gene was examined by the method of Example 1-3 or the method of Example 1-2. At this time, actin-specific primary antibody (Santa Cruz Biotechnology, Germany) was used as an internal control group for protein amount investigation.

As a result, as shown in FIG. 5B, the apoptosis rate by the inhibitor and siRNA was proportional to the decrease in the expression of Erk1 / 2 protein and Rex-1 gene.

That is, BCNU-resistant glioblastoma tumor stem cells had high Rex-1 gene expression and were effectively reduced by Rex-1 siRNA expression, resulting in decreased cell survival due to decreased Ekr1 / 2 activity. Thus, it is predicted that the Rex-1 gene maintains the cellular proliferative capacity of BCNU-resistant tumor stem cells present in glioblastomas U87MG and A172 by inducing Erk1 / 2 activity.

<Example 6> in vivo Confirm tumor suppression

<6-1> Experimental Animal Preparation

Female nude mice (NOD / SCID mice, 5 weeks old) were fed from Charles River Laboratories (USA) and purified in an animal room for 1 week, housed in polycarbonate cages for mice during the acclimation and testing periods. All animal experiments were conducted in accordance with the "Seoul National University Animal Experiment Ethics Regulations".

<6-2> Rex1 siRNA Treatment Performance and Effect Analysis

To examine the effectiveness of Rex1 siRNA treatment, U87MG cells (2 × 10 ⁶ cells / 20 μl DMEM) were mixed with Matrigel (BD Bioscience, USA) and injected subcutaneously in the back of female nude mice. Two weeks after injection, when the diameter of the tumor reached 0.1-0.3 cm, 9 μg of Rex-1 siRNA was dissolved in 10 μl of DMEM and intratumoral injection was performed every three days. Then, the growth of the tumor and the survival rate of the mouse was observed.

As a result, as shown in FIG. 6A, tumors that were not injected with Rex-1 siRNA continued to grow, whereas growth of tumors that were injected with Rex-1 siRNA was slow. In addition, as shown in Figure 6b it was observed that the survival of mice injected with Rex-1 siRNA intratumorally.

<6-3> Histopathological Observation

After 6 weeks of post-intratumoral injection of Rex1 siRNA, tumor tissue was extracted for histological analysis, fixed with 4% paraformaldehyde, and used as a Tissue-Tek optimum cutting temperature (OCT) Compound. ; Sakura Finetek, USA] and frozen at -20 ° C. Tumor tissue was frozen into 10 μm using a freezing microtome (CM3050, Leika Microsystems, German) and transferred to poly-D-lysine-coated slides (Sigma, USA) and dried overnight at 37 ° C. . Histopathological observations were performed by staining with hematoxylin and eosin (eosin; Sigma, USA).

As a result, as shown in Figure 6c the tumor - derived tumor tissue injected with intratumoral Rex-1 siRNA was able to observe a significant number of cell destruction.

<6-4> Sub-Signal Transmission System

<6-4-1> Investigation of protein expression related to self-killing

For tumor tissues of Example 6-2, Bax (1: 1000, Santa Cruz, USA), Bcl-2 (1: 1000, Santa Cruz Biotechnology, Germany), cytochrome C (1: 1000, Santa Cruz Biotechnology, Germany), segmental caspase-3 (Cell Signaling Technology, USA) and PARP (Cell Signaling Technology, USA), etc. using the primary antibody of the protein associated with self-killing expression of the protein of Example 1-3 The method was investigated.

As a result, as shown in Figure 6d it was observed that the protein expression associated with the self-killing is significantly increased in the tumor tissue of mice injected with Rex-1 siRNA.

<6-4-2> Expression of Stem Gene Expression

In the tumor tissue of Example 6-2, the expression level of the stem gene was examined by the method of Example 1-2 using the primer pairs of Table 2.

As a result, as shown in Figure 6e, the expression of stem genes in tumors injected with Rex-1 siRNA was reduced.

<6-4-3> Expression of Neural Stem Cell Markers

In the tumor tissue of Example 6-2, the expression level of the protein was examined by the method of Example 1-3 using Rex-1, the primary antibody of the neural stem cell marker Nestin and GFAP protein.

As a result, as shown in FIG. 6F, GFAP was induced and Rex-1 and Nestin proteins were significantly reduced.

1 is a diagram confirming BCNU resistance of human glioblastoma multiforme cells:

a: BCNU resistance survival rate;

b: gene expression associated with drug transporters;

c: Nestin, a neural stem cell (NSC) marker, and GFAP protein expression (western blot), an astrocyte marker;

d: Nestin and GFAP protein expression (immunochemical analysis); And,

e: Cell migration assay of BCNU-resistant glioblastoma.

2 is a diagram confirming Rex-1 expression of BCNU-resistant glioblastoma:

a: RT-PCR; And,

b: Western blot.

3 is a diagram confirming the effect of the targeted therapy of the Rex-1 gene:

a: inhibition of cell viability of glioblastoma multiforme by treatment with Rex-1 siRNA (phase contrast microscopy);

b: Inhibition of mRNA expression level of Rex-1 of glioblastoma multiforme by treatment with Rex-1 siRNA; And,

c: Inhibition of cell viability of glioblastoma multiforme by treatment with Rex-1 siRNA.

4 is a diagram confirming the inhibition of cell growth and survival signals:

a: specific expression pattern of stem gene of glioblastoma multiforme by treatment with Rex-1 siRNA (Western blot);

b: increased caspase-3 activity of glioblastoma multiforme by treatment with Rex-1 siRNA; And,

c: Expression pattern of MAPKs related protein of glioblastoma multiforme by treatment with Rex-1 siRNA.

5 is a diagram confirming cell survival signal transmission system interference confirmation:

a: reduction of cell viability by Erk1 / 2 inhibitors; And,

b: Reduction of Erk1 / 2 protein and Rex-1 gene expression by Erk1 / 2 inhibitor and Rex-1 siRNA combination.

6 is a diagram confirming the effect of in vivo Rex1 siRNA treatment:

a: tumor growth inhibition;

b: prolong mouse survival;

c: cell destruction in tumor tissue;

d: increased protein expression associated with autokilling;

e: decreased expression of stem genes; And,

f: GFAP induction and decreased Rex-1 and nestin protein expression.

<110> SEOUL NATIONAL UNIVERSITY R & DB Foundation <120> Pharmaceuticla composition comprising siRNA specific for reduced          expression-1 for treating the BCNU-resistance glioblastoma          multiforme <130> 8P-12-05 <160> 19 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ABCG2 forward primer <400> 1 caggaggcct tgggatactt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ABCG2 reverse primer <400> 2 gctatagagg cctggggatt 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TAP1 forward primer <400> 3 gggctgtaag cagtgggaac c 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TAP1 reverse primer <400> 4 caaggccctc caagtgtaag gg 22 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SLC4AZ forward primer <400> 5 tgactgcccc agaaaagag 19 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SLC4AZ reverse primer <400> 6 atccccgata actatyggag ag 22 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 7 catgaccaca gtccatgcca tcact 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 8 tgaggtccac caccctgttg ctgta 25 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rex1 forward primer <400> 9 tgaaagccca catcctaacg 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Rex1 reverse primer <400> 10 caagctatcc tcctgctttg g 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oct4 forward primer <400> 11 cgcacaactg gcattgtcat 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oct4 reverse primer <400> 12 ttctccttga tgtcacgcac 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox2 forward primer <400> 13 tacctcttcc tcccactcca 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox2 reverse primer <400> 14 actctcctct tttgcacccc 20 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NaNog forward primer <400> 15 gctgagatgc ctcacacgga g 21 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NaNog reverse primer <400> 16 tctgtttctt gactgggacc ttgtc 25 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> siRNA target sequence 1 of Rex-1 <400> 17 gaagatggga agcgccaag 19 <210> 18 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> siRNA target sequence 2 of Rex-1 <400> 18 caggggguug ugguuaagcu cuu 23 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> scrambled siRNA <400> 19 ttctccgaac gtgtcacgt 19  

Claims (19)

A pharmaceutical composition for treating BCNU (1,3-Bis (2-chloroethyl) -1-nitrosourea, Carmustine) -resistant glioblastoma multiforme comprising siRNA specific to the target sequence of Rex-1 as an active ingredient. The pharmaceutical composition according to claim 1, wherein the target sequence of Rex-1 is SEQ ID NO: 17 or SEQ ID NO: 18. The single RNA of claim 1, wherein the siRNA comprises an independent sense RNA strand homologous to the target sequence and an antisense RNA strand complementary thereto, or wherein the sense RNA strand and the antisense RNA strand are connected by a loop. Pharmaceutical composition, characterized in that the strand. The pharmaceutical composition of claim 3, wherein the sense RNA strand is SEQ ID NO: 17 or SEQ ID NO: 18. A pharmaceutical composition for treating BCNU-resistant polymorphic glioblastoma, comprising siRNA and BCNU specific for a target sequence of Rex-1. The pharmaceutical composition according to claim 5, wherein the target sequence of Rex-1 is SEQ ID NO: 17 or SEQ ID NO: 18. The single RNA of claim 5, wherein the siRNA comprises an independent sense RNA strand homologous to the target sequence and an antisense RNA strand complementary thereto, or wherein the sense RNA strand and the antisense RNA strand are connected by a loop. Pharmaceutical composition, characterized in that the strand. 8. The pharmaceutical composition of claim 7, wherein the sense RNA strand is SEQ ID NO: 17 or SEQ ID NO: 18. A pharmaceutical composition for treating BCNU-resistant polymorphic glioblastoma, comprising siRNA, BCNU and Erk1 / 2 inhibitors specific for the target sequence of Rex-1. The pharmaceutical composition of claim 9, wherein the target sequence of Rex-1 is SEQ ID NO: 17 or SEQ ID NO: 18. The single RNA of claim 9, wherein the siRNA comprises an independent sense RNA strand homologous to the target sequence and an antisense RNA strand complementary thereto, or wherein the sense RNA strand and the antisense RNA strand are linked by a loop. Pharmaceutical composition, characterized in that the strand. 12. The pharmaceutical composition of claim 11, wherein the sense RNA strand is SEQ ID NO: 17 or SEQ ID NO: 18. The pharmaceutical composition of claim 9, wherein the Erk1 / 2 inhibitor is PD98059 or AG1478. delete delete delete delete delete delete

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