WO2010076935A1

WO2010076935A1 - Pharmaceutical composition comprising sirna specific for reduced expression-1 for treating the bcnu-resistance glioblastoma multiforme

Info

Publication number: WO2010076935A1
Application number: PCT/KR2009/004169
Authority: WO
Inventors: Soo-Kyung Kang
Original assignee: Snu R&Db Foundation
Priority date: 2008-12-31
Filing date: 2009-07-27
Publication date: 2010-07-08
Also published as: KR101087614B1; KR20100080098A

Abstract

The present invention relates to a pharmaceutical composition for treating BCNU(1,3-Bis(2-chloroethyl)-1-nitrosourea, Carmustine)-resistant glioblastoma multiforme, precisely a pharmaceutical composition comprising Rex-1 specific siRNA that is able to inhibit the expression of the stem cell marker Rex-1 (reduced expression-1) for inducing apoptosis of BCNU-resistant glioblastoma multiforme cells and for inhibiting tumor growth. The pharmaceutical composition of the present invention can be effectively used for the treatment of BCNU-resistant glioblastoma multiforme by co-administration with BCNU.

Description

PHARMACEUTICAL 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)-resistance glioblastoma multiforme.

In the past few years, it has been reported that cancers tend to harbor small cell populations with the capability to sustain tumor formation and growth in tumor cells. These cells are referred to as cancer stem cells (CSCs). These CSCs shared many properties with normal stem cells including self-renewal and multi-potency, and expressed a wide variety of transporters involving drug efflux. Recently, several studies identified that several genetic stem cell markers such as Oct4, Nango, and Rex-1 (zfp42) are expressed in various types of cancers such as breast, ovarian, lung, and renal carcinoma. Therefore, developmental approaches to specifically target CSCs by applying the principles of stem cell biology might raise hope to cure the malignant cancer. However, although Oct4, Nango, and Rex-1 (zfp42) are possible markers of CSCs, their functions in CSCs remain unclear.

Among identified genetic stem cell markers, the Rex-1 (for reduced expression-1), a member of the zinc finger protein-42 (zfp-42) family of transcript factors, was first identified as a gene that expresses in F9 embryonal carcinoma (EC) cells. The Rex-1 gene is a well recognized marker for the pluripotent stat of both human and mouse ES cells. It has been reported that targeted deletion of Rex-1 results in loss of ability of endodermal differentiation in F9 EC cells, and that gene silencing by RNA interference for Rex-1 results in loss of capacity of self-renewal in ES cells. Therefore, the Rex-1 expression plays an important role in controlling not only the maintenance of ES cell pluripotency but also the differentiation potential. In addition, Rex-1 has been used as an important pluripotent marker of various stem cells such as multipotent adult progenitor cells and amniotic fluid cells. Moreover, recent investigations indicate that Rex-1 is expressed in human renal cell carcinoma (RCC) implicating Rex-1 as a possible marker of stem cell of RCC. In the point of CSCs view, targeting therapy for Rex-1 gene may be useful for the treatment of cancers.

As compared with other brain tumors, glioblastoma multiforme (GBM) is a relatively aggressive variant in human, which if untreated, leads to a fatal outcome in only a few weeks. Therefore, surgical resection of the primary tumor followed by irradiation and chemotherapy is a palliative measure. BCNU (1,3-Bis(2-chloroethyl)-1-nitrosourea, Carmustine) is the most commonly used chemotherapeutic agent in human GBM because it passes across the blood-brain barrier and the treatment of BCNU might result in the carbamoylation and alkylation of cross-linking DNA strands, thereby causing apoptosis of brain tumor. However, GBM has shown resistance to BCNU chemotherapy, so BCNU chemotherapy fails to substantially prolong median survival of GBM patients. Moreover, clinical studies of GBM have shown that BCNU chemotherapy improves survival compared with radiation treatment alone. However, GBM has shown resistance to BCNU chemotherapy due to variation in multidrug resistance genes, DNA repair activity, and glutathione S-transferase and intracellular glutathione content, resulting in subsequent tumor growth. Recently, several reports have indicated that cancer stem cells (CSCs) could initiate tumors existing in GBM, and these CSCs have drug-resistant capacity. Therefore, temozolomide (TMZ) chemotherapy has provided a new approach for BCNU-resistant brain tumor patients because TMZ enhanced mismatch repair (MMR) triggered by O(6)-alkylguamine during the development of BCNU-resistance. However, although temozolomide significantly increases the proportion of patients surviving for >2 years, long-term survivors are still rare. Moreover, GBM containing CSCs has been found to correlate with the high expression of multidrug resistance (MDR) related genes. Therefore, a new approach to cancer therapy focuses on specific targeting of drug-resistant CSCs populations. The present inventors recently reported that the inhibition of chloride (Cl^-) channel efficiently promotes apoptosis and sensitization to BCNU chemotherapy in GBM CSCs (Kang MK & Kang SK, Biochem Biophys Res Commun. 373(4):539-544, 2008).

The present inventors tried combined-therapy using Rex-1 gene specific siRNA (small interfering RNA) and BCNU in vitro and in vivo to inhibit Rex-1 gene expression in BCNU-resistant glioblastoma where over-expression of Rex-1 mRNA and protein was confirmed. As a result, the present inventors completed this invention by confirming that inhibition of Rex-1 gene expression was effective in inducing apoptosis and inhibiting tumor growth in GBM CSCs.

It is an object of the present invention to provide a pharmaceutical composition for treating BCNU(1,3-Bis(2-chloroethyl)-1-nitrosourea, Carmustine)-resistant glioblastoma multiforme.

It is another object of the present invention to provide a method for treating BCNU-resistant glioblastoma multiform.

To achieve the above objects, the present invention provides a Rex-1 specific siRNA constructed to be working specifically on BCNU-resistant glioblastoma multiforme and a pharmaceutical composition comprising the same as an active ingredient for treating BCNU-resistant glioblastoma multiforme.

The present invention also provides a method for treating BCNU-resistant glioblastoma multiforme using the said siRNA.

The pharmaceutical composition of the present invention can be effectively used for the treatment of BCNU-resistant glioblastoma multiforme by co-administering it with BCNU.

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Figure 1 Figure 5 are diagrams illustrating the BCNU-resistance of human glioblastoma multiforme cells:

Figure 1: BCNU resistance and cell viability;

Figure 2: expressions of genes involved in drug delivery;

Figure 3: Expressions of Nestin (neural stem cell marker) and GFAP (astrocyte cell marker) (Western blot analysis);

Figure 4: Expressions of Nestin and GFAP (immunochemical analysis); and,

Figure 5: migration of BCNU-resistant glioblastoma cells.

Figure 6 Figure 7 are diagrams illustrating the expression of Rex-1 in BCNU-resistant glioblastoma multiforme:

Figure 6: RT-PCR; and,

Figure 7: Western blot.

Figure 8 Figure 10 are diagrams confirming the effect of Rex-1 gene target therapy:

Figure 8: inhibition of viability of glioblastoma multiforme cells by the treatment of Rex-1 siRNA (observed by phase contrast microscope);

Figure 9: inhibition of Rex-1 mRNA expression in glioblastoma multiforme by the treatment of Rex-1 siRNA; and

Figure 10: inhibition of viability of glioblastoma multiforme cells by the treatment of Rex-1 siRNA

Figure 11 Figure 13 are diagrams confirming the inhibition of cell growth and cell survival signal:

Figure 11: specific expression pattern of stemness gene of glioblastoma multiforme by the treatment of Rex-1 siRNA (Western blot);

Figure 12: increase of caspase-3 activity in glioblastoma multiforme by the treatment of Rex-1 siRNA; and

Figure 13: expression patterns of MAPKs related proteins in glioblastoma multiforme by the treatment of Rex-1 siRNA.

Figure 14- Figure 15 are diagrams confirming the interference of cell survival signal pathway:

Figure 14: decrease of cell viability by the treatment of Erk1/2 inhibitor; and

Figure 15: down-regulation of Erk1/2 protein and Rex-1 gene by the co-treatment of Erk1/2 inhibitor and Rex-1 siRNA.

Figure 16 - Figure 21 are diagrams confirming the effect of in vivo Rex-1 siRNA treatment:

Figure 16: inhibition of tumor growth;

Figure 17: increase of mouse survival rate;

Figure 18: cellular disruption in tumor tissue;

Figure 19: increase of apoptosis related protein expression;

Figure 20: decrease of stemness gene expression; and

Figure 21: induction of GFAP, and decreases of Rex-1 and Nestin expressions.

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

The present invention also provides a pharmaceutical composition comprising Rex-1 target sequence specific siRNA and BCNU as active ingredients for treating BCNU-resistant glioblastoma multiforme.

The present invention further provides a method for treating BCNU-resistant glioblastoma multiforme containing the step of administering Rex-1 target sequence specific siRNA to a subject with BCNU-resistant glioblastoma multiforme.

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

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

Terms used in this description are defined hereinafter.

The term siRNA indicates double-stranded RNA capable of inducing RNA interference (RNAi) by cleavage of target gene mRNA and is composed of sense RNA strand having the homologous sequence with the target gene mRNA and antisense RNA strand having the complementary sequence to the sequence. Since the siRNA can inhibit the target gene expression, it can be effective for gene knock-down or gene therapy.

The phrase "inhibition of gene expression" indicates the capability of silencing of Rex-1 gene. Inhibition of the Rex-1 gene expression can be achieved when the test value is approximately 90%, preferably 50% and more preferably 25 - 0% by the value of the control.

Analysis can be properly performed by those methods well known to those in the art, for example, dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function and phenotype assay based methods at protein or mRNA level.

The term "specific" indicates the power that can only inhibit a target gene without affecting any other genes in cells and particularly in this invention, the term indicates Rex-1 specific.

Hereinafter, the present invention is described in detail.

As confirmed in an example of the present invention, when U87MG and A172 cells (glioblastoma cells) were treated with 33 ㎍/㎖ of BCNU, the cell viability was significantly decreased and only a few cells survived. In the case of 33 ㎍/㎖ BCNU treatment for 3 days, U87MG and A172 cells had a low survival rate of 22.45 % and 29.03 %, respectively (see Figure 1). Additionally, these survived glioblastoma cells highly expressed several genes related to drug transporters such as ABCG2, TAP1, and SLCA42A (see Figure 2). These results indicated that Nestin-positive subpopulations of U87MG and A172 were resistant and survived after BCNU chemotherapy.

These survived U87MG and A172 cells showed high immunoreactivity to Nestin, the marker for the neural stem cells (NSC) and low immunoreactivity to GFAP, the marker of astrocyte (see Figure 3). Moreover, these BCNU-resistant U87MG and A172 cells expressed GFAP while the Nestin was lost during in vitro differentiation (see Figure 4), indicating that BCNU-resistant GBM cells are committed to produce a progeny of differentiated cells with phenotypic features of glial cells.

Furthermore, the BCNU-resistant cells derived from U87MG and A172 were significantly increased the migration efficiencies as compared to control cells (see Figure 5). This finding showed that BCNU-resistant cells derived from U87MG and A172 are CSCs with multipotency abilities as shown in our previous studies

To further evaluate the CSCs properties of BCNU-resistant glioma cells, the present inventors determined the distinctive expression patterns of several stemness genes including Rex-1, Nanog, Sox2 and Oct4, have been identified as potential markers of human embryonic and adult stem cells. As a result, mRNA and protein of Rex1, Nango, Sox2 and Oct4 were overexpressed in the BCNU-resistant GBM cells compared with control cells (see Figures 6 and 7). Although the stemness genes and proteins were constitutively expressed in control glioblastoma cells, their expression levels were increased in BCNU-resistant GBM subpopulations. Rex-1 expression, in particular, was markedly elevated in BCNU-resistant cells derived from U87MG and A172. This result is further supported in our previous study that BCNU-resistant subpopulations derived from GBM have CSCs properties

To gain insight between the roles of Rex-1 and chemotherapeutic drug-induced resistance in the CSCs of glioblastoma, the U87MG and A172 cells were transfected with Rex-1 silencing RNA (siRNA) prior to BCNU treatment. Rex-1 siRNA-transfected GBM cells were synergistically inhibited the cell survival (see Figure 8). And the expression of Rex-1 and the cell survival were inhibited by Rex1 siRNA/BCNU combined-therapy (see Figures 9 and 10). Therefore, Rex-1 gene silencing by RNA interference resulted in loss of capacity to self-renew in ES cell. In human GBM cell lines, U87MG and A172, Rex-1 siRNA/BCNU combined-therapy could effectively block BCNU-resistant CSCs survivals in vitro.

In a preferred embodiment of the present invention, growth inhibition pattern of glioblastoma multiforme by Rex-1 siRNA was investigated. To do so, the expressions of apoptosis related proteins such as Bax, Bcl-2, cytochrome C, P53, P21 and c-myc were measured after the Rex-1 siRNA/BCNU combined-therapy. As a result, the expression of Bcl-2 protein was reduced, but the expressions of Bax, cytochrome C and c-myc related p53 and p21 were increased by Rex-1 gene silencing, leading to apoptosis (see Figure 11). The activity of caspase-3 was also significantly increased in U87MG and A172 cells at by 1.8- and 1.9-fold, respectively, by Rex-1 siRNA/BCNU combined-therapy (see Figure 12). These results suggested that direct inhibition of Rex-1 by siRNA increases the sensitivity for BCNU chemotherapy and results in an intense apoptosis of GBM cells.

During apoptosis, differential activation of MAPKs (Mitogen-Activated Protein Kinases) has been reported in many instances; the proapoptotic function of JNK (Jun kinase) and p38 MAPK, and antiapoptotic function role of Erk were reported. In a preferred embodiment of the present invention, the inventors performed Western blot analysis to further explain the mechanisms underlying the apoptosis induce by Rex-1 siRNA/BCNU combined-therapy. As a result, when two types of GBM cells were combined-treated with Rex-1 siRNA and BCNU, it was observed that the phosporylation of Erk1/2 was significantly inhibited in both GBM cells. In contrast, the activation of JNK and p38 were differentially phosphoylated by Rex-1 siRNA/BCNU combined-therapy in U87MG and A172 cells (see Figure 13). From these results, it was confirmed that Rex-1 siRNA/BCNU combined-therapy-induced apoptosis is correlated with the inactivation of Erk1/2 in GBM cells although p38 and JNK are differentially activated in cell-type specific.

To further assess the effect of Rex-1 siRNA on Erk1/2 inhibition and growth inhibition on GBM cells, the present inventors evaluated the cell survival and Rex1 expression pattern after the treatment on BCNU-resistant CSCs derived from U87MG and A172 with PD98059 (specific inhibitor of Erk1/2), AG1478 (broad range inhibitor of Erk1/2), or Rex-1 siRNA, respectively. As a result, PD098059 and AG1478 significantly attenuated cell survival in GBM cell populations (see Figure 14). AG1478 was more effective in targeting of BCNU-resistant glioblastoma CSCs compared with PD98059. Particularly, on 3 days post-Rex-1 siRNA transfection, almost glioblastoma CSCs population was induced cell death (see Figure 14). Furthermore, the rate of cell death by inhibitors and siRNA was proportional to the decrease in the activations of Erk1/2 and Rex-1 (see Figure 15). Collectively, BCNU-resistant GBM CSCs expressed Rex-1 at a high level, which was successfully reduced by Rex-1 siRNA, and finally resulted in the inhibition of cell survival by the reduction of Erk1/2 activation. Therefore, it is also suggested that Rex-1 might maintain the cell proliferation potential of BCNU-resistant CSCs in U87MG and A172 by promoting Erk1/2 activity.

In a preferred embodiment of the present invention, it was investigated whether in vitro results for functional growth inhibition by Rex-1 gene targeting were able to effect a change in vivo. As a result, the mice non-treated with Rex-1 siRNA showed progressive tumor growth, whereas the mice treated with Rex-1 siRNA showed delayed tumor growth (see Figure 16). And Rex-1 siRNA intratumoral injection significantly prolonged the survival of the treated mice (see Figure 17).

To investigate the role of Rex-1 siRNA in the in vivo xenograft tumor growth, the present inventors performed histopathological examination and determined downstream signaling molecules. Histopathologically, the tumor tissue sections derived from intratumoral Rex-1 siRNA-injected mice showed significant higher cellular disruption (see Figure 18). The expression of apoptosis-related proteins such as Bax, cytochrome C, and cleavage form of caspase-3 and PARP were also significantly increased in intratumoral Rex-1 siRNA-injected tumor tissues (see Figure 19). Moreover, reduction in the expression of stemness genes was observed in Rex-1 siRNA treated-tumor (see Figure 20). Our data also showed induction of GFAP and drastic reduction of Rex-1 and Nestin in tumors by Rex-1 siRNA-intratumoral injection (see Figure 21). These results demonstrated that lack of Rex-1 expression resulted in reduction of cell survival and induction of apoptosis on CSCs expressing Nestin and stemness gene in tumor that ultimately suppressed the growth of glioblastoma. Collectively, it was confirmed that Rex-1 siRNA could successfully reduce CSCs in vivo and thereby markedly retard tumor growth.

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

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

The said Rex-1 target sequence is characteristically the sequence represented by SEQ. ID. NO: 17 or NO: 18. The said siRNA contains an independent sense RNA strand which is homologous to the said target sequence or a complementary antisense RNA strand or can be single-stranded RNA having stem-loop structure where the sense RNA and the antisense RNA are linked by loop. The sense RNA strand herein is preferably the sequence represented by SEQ. ID. NO: 17 or NO: 18, but not always limited thereto.

The said siRNA is double-stranded RNA having a complete RNA pair, but not always limited thereto and can have hairpin structure of stem-loop, which is called shRNA (short hairpin RNA). The double-stranded chain or the stem can also contain non-paired region of mismatch (having no complementary bases) or bulge (having no corresponding bases). The total length of it is preferably 10 - 80 bp, preferably 15 - 60 bp and more preferably 20 - 40 bp. The loop is not limited to a specific sequence and only requires 3 - 10 bp in order to link the sense sequence and the antisense sequence properly. The conventional siRNA loops are exemplified 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 of www.genscript.com). The said siRNA can have blunt end or cohesive end. The cohesive end herein can have 3 end protruding structure or 5 end protruding structure and at this time the numbers of protruding bases are not limited. For example, 1 - 8 bases, preferably 2 - 6 bases can be protruded. The said siRNA can include low-molecular RNA (for example, natural RNA molecules such as tRNA, rRNA and viral RNA or artificial RNA molecules) in one protruding end, as long as the target gene inhibition activity is still in effect. Both ends of the siRNA are not necessarily all cleavage ends. One end of one of the double-stranded RNA can be stem-loop structure linked by a linker RNA. The length of a linker is not limited as long as it is capable of linking a pair in stem.

The said siRNA is generally administered as a pharmaceutical composition. The administration can be performed in vitro or in vivo by any known method that can introduce nucleic acid into a target cell. A method for intracellular introduction of siRNA is not limited. The siRNA can be synthesized and then directly inserted into a host cell. Or siRNA expression vector designed to express the siRNA in a cell or PCR-based siRNA expression cassette can be introduced into a cell by transfection or infection. And at this time, the expression vector is not limited and any conventional siRNA expression vector that is able to express a target sequence as siRNA in a cell can be used. The vector is exemplified by GeneClip U1 Hairpin Cloning System (Cat.#'s C8750, C8760, C8770, C8780, C8790), siLentGene U6 Cassette RNA Interference System (Cat.# C7800), 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) of Promega and pRNA-U6.1 (Cat.#'s SD1201, SD1202, SD1207), pRNATin-H1.2 (Cat.#'s SD1223, 1224) and pRNA-H1.1/Adeno (Cat.# SD12009) of Genscrpit.

A general method for gene introduction into a target cell is exemplified by calcium phosphate, DEAE-dextran, electroporation, microinjection and virus method (Graham, F.L. and van der Eb, A.J. (1973) Virol. 52, 456; McCutchan, J.H. and Pagano, J.S. (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, M.R. (1980), Cell 22, 479). Cationic liposome method is recent addition to such methods introducing nucleotide into a cell (Felgner, P.L., et al., (1987), Proc. Nati. Acad. Sci. USA 84, 7413). Commercial cationic liposome is exemplified by Tfx 50 (Promega) or Lipofectamin 2000 (Life Technologies), etc. A method to directly insert siRNA that is able to be xenografted complementarily to Rex-1 gene is not limited, but preferably performed as follows: 2 6 ㎍ of cationic liposome is mixed with 1 ㎍ of siRNA, which proceeds to lipofection for 15 - 40 minutes.

The said pharmaceutical composition can be administered by any acceptable methods such as injection, oral administration, topical administration, nasal administration, and rectal application, etc. The carrier herein is selected among any acceptable pharmaceutical carriers, which is preferably exemplified by liposome, particularly cationic liposome. And more preferable administration method is injection.

The pharmaceutical composition can be used as a therapeutic agent for human or animals and the pharmaceutical composition herein can be formulated in the forms of injectable solutions, creams, ointments, tablets, suspensions, etc.

The pharmaceutical composition of the present invention can additionally contain a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable excipient is well known to those in the art and is a comparatively inactive material that facilitates administration of a pharmaceutically active material. For example, the excipient can give shape or viscosity or plays a role as a diluent. A preferred excipient is exemplified by a stabilizer, a moisturizer, an emulsifier, salts capable of changing osmolarity, an encapsulating agent, a buffer, and a skin diffusion promoter, but not always limited thereto. The excipient for carrying a drug via oral or parenteral administration and formulation thereof are described in Remington, The Science and Practice of Pharmnacy (the 20^th edition), Mack Publishing (2000).

Effective dose of the siRNA is determined to reduce Rex-1 expression significantly enough, compared with the expression level in normal condition in the absence of siRNA. The siRNA can be introduced by the amount that can deliver at least one copy per cell. The higher the level of the double-stranded material (ex. at least 5, 10, 100, 500 or 1000 copies per cell), the higher the inhibitory effect is. But, in some cases, small copies might be preferred.

In a preferred embodiment of the present invention, the expression of Rex-1 and the cell survival were inhibited by Rex1 siRNA/BCNU combined-therapy (see Figures 9 and 10). Therefore, Rex-1 gene silencing by RNA interference resulted in loss of capacity to self-renew in ES cell. Rex-1 siRNA/BCNU combined-therapy could effectively block BCNU-resistant CSCs survivals in vitro. So, the combined-therapy of Rex-1 target sequence specific siRNA and BCNU can be an effective method for treating BCNU-resistant glioblastoma multiforme.

In a preferred embodiment of the present invention, Rex-1 siRNA-transfected GBM cells were synergistically inhibited the cell survival (see Figure 8). That is, Rex-1 gene silencing by RNA interference resulted in loss of capacity to self-renew in ES cell and effectively inhibited in vitro survival of BCNU-resistant CSCs. So, the Rex-1 target sequence specific siRNA of the present invention can be effectively used for the treatment of BCNU-resistant glioblastoma multiforme.

The BCNU-resistant glioblastoma multiforme can be isolated or cultured cells expressing Rex-1 gene or can be included in mammals without being isolated. The mammals herein indicate human and non-human mammals, for example dog, cat, horse, pig, sheep, cow, goat, rodents such as hamster, mouse and rat, and apes. In this invention, U87UG and A172 were used as the BCNU-resistant glioblastoma multiforme expressing the target gene.

In this invention, administration indicates a way to introduce a required amount of material into a patient via proper pathway and at this time the pathway for delivering the material can be any general pathway facilitating delivery of a target material to a target place, which is herein exemplified by intraperitoneal administration, intravenous administration, intramuscular administration, hypodermic administration, intradermal administration, oral administration, local administration, intranasal administration, intrapulmonary administration, and intrarectal administration, but not always limited thereto. The pharmaceutical composition can be administered by a random device facilitating delivery of an active ingredient to a target cell.

The term "administration" herein also indicates the introduction of the siRNA of the present invention in the cell expressing Rex-1 gene by "systemic transfer" or "local transfer". "systemic transfer" indicates transfer inducing broad bio-distribution of a compound in an organism. Some techniques facilitate systemic transfer of a specific compound and others do not. Again, systemic transfer means that a therapeutically effective dose of a compound is exposed to a whole body. In general, to achieve broad bio-distribution, it is important for a compound to have a required half-life in blood in order for the compound not to be decomposed or eliminated before it travels far from the administration site (for example, not to be decomposed by the first transit organ such as liver or lung or by non-specific cellular interaction). Systemic transfer of nucleic acid-lipid can be achieved by a random pathway selected among those well-known to those in the art, for example intravenous administration, hypodermic administration or intraperitoneal administration, etc. In a preferred embodiment of the present invention, nucleic acid is widely distributed over whole body by intravenous administration. In this description, "local transfer" indicates a direct delivery of a compound to a target area. For example, a compound is directly injected into a tumor or any other target area, for example inflammation region or target organs such as liver, heart, pancreas, and kidney.

The present invention provides a method for treating BCNU-resistant glioblastoma multiforme containing the step of co-administering the Rex-1 target sequence specific siRNA of the present invention and BCNU to a subject with BCNU-resistant glioblastoma multiforme.

For the "co-administration" siRNA is treated a couple of hours before BCNU treatment.

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

The Erk1/2 inhibitor herein is the Erk1/2 specific inhibitor PD98059 or the Erk1/2 broad range inhibitor AG1478.

In a preferred embodiment of the present invention, the present inventors evaluated the cell survival and Rex1 expression pattern after the treatment on BCNU-resistant CSCs with PD98059 (specific inhibitor of Erk1/2), AG1478 (broad range inhibitor of Erk1/2), or Rex-1 siRNA, respectively. As a result, PD098059 and AG1478 attenuated cell survival significantly in GBM cell populations (see Figure 14). Particularly, on 3 days post-Rex-1 siRNA transfection, almost glioblastoma CSCs population was induced cell death (see Figure 14). Furthermore, the rate of cell death by inhibitors and siRNA was proportional to the decrease in the activations of Erk1/2 and Rex-1 (see Figure 15). Collectively, BCNU-resistant GBM CSCs expressed Rex-1 at a high level, which was successfully reduced by Rex-1 siRNA, and finally resulted in the inhibition of cell survival by the reduction of Erk1/2 activation. Therefore, it is also suggested that Rex-1 might maintain the cell proliferation potential of BCNU-resistant CSCs in U87MG and A172 by promoting Erk1/2 activity. So, the combined treatment of the Rex-1 target sequence specific siRNA of the present invention, BCNU and Erk1/2 inhibitor can be effectively used for the treatment of BCNU-resistant glioblastoma multiforme.

In addition, the present invention provides a use of Rex-1 target sequence specific siRNA for the prevention and treatment of BCNU(1,3-Bis(2-chloroethyl)-1-nitrosourea, Carmustine)-resistant glioblastoma multiforme.

As described hereinbefore, the present invention is to develop siRNA to treat BCNU-resistant glioblastoma multiforme by regulating Rex-1 specifically. Particularly, the present invention found out a novel use of siRNA represented by SEQ. ID. NO: 17 or NO: 18. However, the said siRNA is not limited to those sequences represented by SEQ. ID. NO: 17 or NO: 18, and any siRNA that is capable of binding to mRNA encoding Rex-1 can be accepted.

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Confirmation of properties of BCNU-resistant glioblastoma cells

<1-1> BCNU-resistant survival rate of BCNU-resistant glioblastoma cells

Human glioblastoma multiforme cell lines, U87MG and A172 were obtained from American Type Culture Collection (ATCC, USA). The cells were maintained in DMEM (Gibco Laboratories, USA) supplemented with 10% FBS and 100 units/㎖ penicillin-streptomycin. All of the experiments were performed on cultures that were at 70% confluence when the cells were in log phase growth. To determine the drug sensitivity of the cultured cancer cells, U87MG and A172 cells were plated on 24-well plates and treated with BCNU (33 ㎍/㎖; 1,3-bis(2-chloroethyl)-1-nitrosourea, Sigma Chemical Company, USA), and further incubated at 37℃ in an atmosphere with 5% CO₂ for 3 days. The control group was treated with DMSO instead of BCNU. Cell viabilities were determined by the trypan blue dye exclusion method. All experiments were performed in triplicate. All data were expressed as the means standard deviation (SD) from three independent experiments. Statistical analysis was performed by a two-tailed Student's t-test (P<0.05).

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

<1-2> BCNU-resistance of BCNU-resistant glioblastoma cells

Expression patterns of several genes related to drug transporters such as ABCG2, TAP1, and SLCA42A in the survived BCNU-resistant tumor cells of Example <1-1> and the control BCNU-resistant tumor cells were investigated. Total RNA was isolated with trizol (Life Technologies, USA) and was reverse transcribed into first-strand cDNA using oligo-dT primer. Amplification was performed using 20 pM of specific primers (Table 1) as follows: predenaturation at 95℃ for 5 minutes, denaturation at 95℃ for 1 minute, annealing at a proper temperature for 1 minute, polymerization at 72℃ for 1 minute, 35 cycles from denaturation to polymerization, and final extension at 72℃ for 7 minutes. The annealing temperature for each primer varied as follows: ABCG2 54℃, TAP1 60℃, SLC4AZ 55℃, and GAPDH 55℃. Following PCR, 6 ㎕ of the samples were loaded onto 1% agarose gel, electrophoresed, kept in ethidium bromide and visualized under UV light. At this time, GAPDH was used as the internal control.

As a result, as shown in Figure 2, expressions of such genes related to drug transporters including ABCG2, TAP1, and SLCA42A were high in the survived glioblastoma cells.

Table 1

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

<1-3> Cancer stem cell properties of BCNU-resistant glioblastoma cells

For analysis of protein expressions of neuronal and glial markers in the survived BCNU-resistant tumor cells of Example <1-1>, the resistant cells were fixed with 4% paraformaldehyde fixative solution for 30 min at room temperature. After washing with PBS, the cells were incubated with primary antibodies against anti-GFAP (1:1500, DAKO Cytomation, Denmark) and anti-Nestin (1:200, Sigma, USA) overnight at 4℃. After extensive washing with PBS, the cells were incubated for 30 min with FITC, Texas-Red, or TRITC conjugated secondary antibodies (1:250, Molecular Probe, USA). Cell nuclei were labeled with 4-6'diamidino-2-phenylindoline (DAPI; Vector laboratories, UK) and analyzed using Confocal Microscopy (Leica Microsystems, USA). Immunocytochemical experiments were repeated at least three times.

As a result, as shown in Figure 3, the survived U87MG and A172 cells showed high immunoreactivity to Nestin, the marker for the neural stem cells (NSC) and low immunoreactivity to GFAP, the marker of astrocytes.

<1-4> Cancer stem cell properties of BCNU-resistant glioblastoma cells

The survived BCNU-resistant tumor cells of Example <1-1> were lysed and sonicated in PRO-PREP^TM Protein Extraction Solution (iNTRON Biotechnology, Inc., USA). Equivalent amounts (40㎍) of protein were fractionated on 12% SDS-PAGE gels, transferred onto nitrocellulose membranes (Sigma, USA). The membranes were blocked in 5% skim milk (Difco, USA) at room temperature for 2 hours. Antigen-antibody reaction was induced at 4℃ using anti-GFAP (1:1500, DAKO, USA) and anti-Nestin (1:200, Sigma, USA) primary antibodies, followed by reaction with HRP (horseradish peroxidase, Cell Signaling Technology, USA) conjugated secondary antibodies. Protein expression was measured using ECL kit (enhanced chemiluminescence solution kit, Amersham, UK) and comparative protein expression was determined by using Quantify-one 1-D analysis soft-ware. Relative band intensities were determined by Quantity-one 1-D analysis software (Bio-Rad, USA).

As a result, as shown in Figure 4, the survived BCNU-resistant U87MG and A172 cells expressed GFAP while Nestin was lost during in vitro differentiation, indicating that BCNU-resistant GBM cells are committed to produce a progeny of differentiated cells with phenotypic features of glial cells.

<1-5> Cell migration assay with BCNU-resistant glioblastoma cells

The survived BCNU-resistant tumor cells (5x10⁵ cells) of Example <1-1> were transferred into Costar transwell membranes (Corning, USA) and placed in six-well plates. The plate was incubated overnight at 37℃ in a 37℃/CO₂ incubator. The cells migrated on the lower chamber were stained with Harris hematoxylin (Sigma, USA) for 20 minutes, and then washed. The number of cells on the lower chamber was counted under microscope (x 200).

As a result, as shown in Figure 5, the BCNU-resistant cells derived from U87MG and A172 were significantly increased the migration efficiencies, compared with control cells.

Example 2: Rex-1 expression in BCNU-resistant glioblastoma cells

<2-1> Gene expression analysis

To further evaluate the CSCs properties of the survived BCNU-resistant tumor cells of Example <1-1>, the present inventors investigated the distinctive expression patterns of several stemness genes including Rex-1, Nanog, Sox-2 and Oct-4 using primer sets shown in Table 2 by the similar manner as described in Example <1-2>. Annealing temperature for each primer was as follows: Rex1 55℃, Oct4 53℃, Sox2 59℃, Nanog 59℃, and GAPDH 55℃.

As a result, as shown in Figure 6, the BCNU-resistant GBM cells overexpressed mRNA of Rex-1, Nango, Sox-2 and Oct-4 compared with control cells. In particular, Rex-1 expression was markedly elevated in BCNU-resistant cells derived from U87MG and A172.

Table 2

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

<2-2> Protein expression analysis

The distinctive expression patterns of several stemness genes in the survived BCNU-resistant tumor cells of Example <1-1> were investigated by the similar manner as described in Example <1-3> using Rex1 (1:1000, Abcam, UK), Nango (1:1000, Abcam, UK) Sox-2 (1:1000, Chemicon, USA) and Oct4 (1:1000, Santa Cruz Biotechnology, Germany) specific primary antibodies.

As a result, as shown in Figure 7, the BCNU-resistant GBM cells overexpressed proteins of Rex-1, Nango, Sox-2 and Oct-4 compared with control cells. In particular, Rex-1 expression was markedly elevated in BCNU-resistant cells derived from U87MG and A172.

The above results support the previous study that BCNU-resistant subpopulations derived from GBM have CSCs properties.

Example 3: Efficacy of Rex-1 gene target therapy

<3-1> Rex-1 siRNA transfection

To investigate the relation of Rex-1 and chemotherapeutic drug-induced resistance in the BCNU-resistant GBM CSCs, the cells were transfected with Rex-1 siRNA before being treated with BCNU.

Particularly, transfection of siRNA was performed by using LipofectAMINE 2000 (Invitrogen, USA). Human glioblastoma U87MG and A172 cells were cultured in DMEM (complete medium) supplemented with 10% FBS at a confluency of 60-70% in six-well plates. At 24 hr after plating, the complete medium was replaced with serum-free medium, and then transfected with 5 ㎍ of siRNA (SEQ. ID. NO: 17: 5'-GAAGAUGGGAAGCGCCAAG-3'; SEQ. ID. NO: 18: 5'-CAGGGGGUUGUGGUUAAGCUCUU-3', Dharmacon Inc., USA) per well using LipofectAMINE 2000. After 5 h, the serum-free medium was replaced again with complete medium and treated with 33 ㎍/㎖ of BCNU for 48 h at 37℃ in 5% CO₂. A scramble siRNA sequence (SEQ. ID. NO: 19: 5'-UUCUCCGAACGUGUCACGU-3', Gima Biol Engineering Inc., China) was used as the negative control. And as the positive control, BCNU alone was treated thereto.

<3-2> Relation of Rex-1 and chemotherapeutic drug-induced resistance

<3-2-1> Analysis of cell survival rate

Cell survival rate of the cells transfected in Example <3-1> was investigated using phase-contrast microscope (Nikon, Japan).

As a result, as shown in Figure 8, cell survival rate of GBM cells was significantly reduced by the treatment of Rex-1 siRNA.

<3-2-2> Rex-1 expression

The expression of Rex-1 mRNA in the cells transfected in Example <3-1> was investigated by the same manner as described in Example <1-2> by using the primer set comprising sequences each represented by SEQ. ID. NO: 9 and NO: 10.

As a result, as shown in Figure 9, the expression of Rex-1 mRNA in the GBM cells transfected with Rex-1 siRNA was significantly inhibited.

<3-3> Cell survival rate over the duration of treatment

The cells transfected in Example <3-1> were recovered on day 1, day 2, and day 3, followed by the investigation of cell survival rate by the same manner as described in Example <1-1>.

As a result, as shown in Figure 10, cell survival rate of the GBM cells transfected with Rex-1 siRNA was reduced.

Example 4: Inhibition of cell growth and survival signal

<4-1> Inhibition of cell growth

The distinctive expression patterns of several stemness genes in the BCNU-resistant tumor cells transfected in Example <3-1> were investigated using 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 by the same manner as described in Example <1-3>.

As a result, as shown in Figure 11, the expression of Bcl-2 protein was decreased, but the expressions of Bax and cytochrome C protein were up-regulated. Furthermore, the p53 and p21 proteins coupled with c-myc protein were over-expressed by gene silencing of Rex-1.

<4-2> Caspase-3 activity

The transfected cells of Example <3-1> were washed with HBSS (Hank's Balanced Salts, Modified) and lysed in CytoBuster^TM Protein Extraction Reagent (Novagen, USA) for 1 hour at 4 C, followed by centrifugation (12,000 rpm) for 5 minutes. Substrate for caspase-3 (Chelbiochem, Germany) was added in 50 ㎕ reaction buffer. Reaction mixture was then incubated at 37℃ for 2 hours. Optical density was measured at 405㎚ in a micro reader (Molecular Devices, USA).

As a result, as shown in Figure 12, the activity of caspase-3 was significantly increased on U87MG and A172 cells at by 1.8- and 1.9-fold, respectively, by Rex-1 siRNA/BCNU combined-therapy.

<4-3> Mechanism of apoptosis

The expressions of MAPKs (Mitogen-Activated Protein Kinases) related proteins in the BCNU-resistant tumor cells transfected in Example <3-1> were investigated by using Erk1/2 (1:1000, Cell Signaling Technology, USA), p38 (1:1000, Cell Signaling Technology, USA), SAPK/JNK (Stress-activated protein kinase/c-Jun NH2-terminal kinase) (1:1000, Cell Signaling Technology, USA) and GAPDH (internal control) specific primary antibodies by the similar manner as described in Example <3-1>.

As a result, as shown in Figure 13, the phosporylation of Erk1/2 was significantly inhibited in two types of GBM cells. In contrast, the activation of JNK and p38 were differentially phosphorylated in U87MG and A172 cells.

Example 5: Interference of cell survival signal

<5-1> Cell survival rate

The BCNU-resistant tumor cells were treated with 10 uM of the Erk1/2 specific inhibitor PD98059 (Sigma, USA) and the Erk1/2 broad range inhibitor AG1478 (Chemicon, USA) respectively instead of the said transfection. Cell survival rate was investigated 1, 2 and 3 days later by the same manner as described in Example <1-1>.

As a result, as shown in Figure 14, PD098059, the specific inhibitor of Erk1/2, and AG1478, the broad range inhibitor of Erk1/2, attenuated cell survival significantly in GBM cell populations. AG1478 was more effective in targeting of BCNU-resistant glioblastoma CSCs compared with PD98059. In particular, almost glioblastoma CSCs population was induced cell death on 3 days post-Rex-1 siRNA transfection.

<5-2> Expressions of Erk1/2 and Rex-1

The relation of the rate of cell death by inhibitors and siRNA and the expressions of Erk1/2 and Rex-1 gene was investigated by the same manner as described in Example <1-2> or Example <1-2>. As the internal control for quantification of proteins, an actin-specific primary antibody (Santa Cruz Biotechnology, Germany) was used.

As a result, as shown in Figure 15, the rate of cell death by inhibitors and siRNA was proportional to the decrease in the activations of Erk1/2 and Rex-1.

That is, BCNU-resistant GBM CSCs expressed Rex-1 at a high level, which was successfully reduced by Rex-1 siRNA, and finally resulted in the inhibition of cell survival by the reduction of Erk1/2 activation. Therefore, it is also suggested that Rex-1 might maintain the cell proliferation potential of BCNU-resistant CSCs in U87MG and A172 by promoting Erk1/2 activity.

Example 6: Inhibition of in vivo tumorigenesis

<6-1> Test animals

Female nude mice (NOD/SCID mice, 5-week-old females) were purchased from the Charles River Laboratories (USA). The mice were adapted in an animal laboratory for 1 week. The animals were kept in polycarbonate cages for mouse during the adaptation and experimental period. Feeds and water were given freely. The experiments were all conducted in accordance with the institutional guidelines established by the Seoul National University.

<6-2> Evaluation of efficacy of Rex-1 siRNA therapy

To evaluate the efficacy of Rex-1 siRNA therapy, U87MG tumor cells (2 10⁶ cells/20 ㎕ DMEM) were mixed with Matrigel (BD Bioscience, USA) and subcutaneously implanted into female nude mice. After two weeks, when the tumors had reached 0.1 ~ 0.3 ㎝in diameter, 9 ㎍ of Rex-1 siRNA in 10 ㎕ of DMEM was injected intratumorally at three-day intervals. Then, tumor growth and the survival of the treated mice were observed.

As a result, as shown in Figure 16, the mice non-treated with Rex-1 siRNA showed progressive tumor growth, whereas the mice treated with Rex-1 siRNA showed delayed tumor growth. As shown in Figure 17, siRNA against Rex-1 intratumoral injection significantly prolonged the survival of the treated mice.

<6-3> Histopathological examination

The mice were sacrificed at 6 weeks post-intratumoral injection of Rex-1 siRNA and tumor tissues were extracted. In order to assess histologic analysis, the tumors were fixed with 4% paraformaldehyde and embedded in Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Finetek, USA) and then frozen at -20℃. Cryostat sections of the tumor (10㎛) were cut using a freezing microtome (CM3050, Leika Microsystems, Germany). The sections were mounted on poly-D-lysine-coated slides (Sigma, USA) and air-dried overnight at 37℃. The sections were stained with hematoxylin and eosin (Sigma, USA) and observed under optical microscope for histopathological examination.

As a result, as shown in Figure 18, the tumor tissue sections derived from intratumoral Rex-1 siRNA-injected mice showed significant higher cellular disruption.

<6-4> Downstream signaling molecules

<6-4-1> Expressions of apoptosis-related proteins

Expressions of apoptosis-related proteins in the tumor tissues of Example <6-2> were investigated using Bax (1:1000, Santa Cruz, USA), Bcl-2 (1:1000, Santa Cruz Biotechnology, Germany), cytochrome C (1:1000, Santa Cruz Biotechnology, Germany), cleavage form of caspase-3 (Cell Signaling Technology, USA) and PARP (Cell Signaling Technology, USA) primary antibodies by the same manner as described in Example <1-3>.

As a result, as shown in Figure 19, the expressions of apoptosis-related proteins were also significantly increased in intratumoral Rex-1 siRNA-injected tumor tissues.

<6-4-2> Expressions of stemness genes

Expressions of stemness genes in the tumor tissues of Example <6-2> were investigated using the primer sets shown in Table 2 by the same manner as described in Example <1-2>.

As a result, as shown in Figure 20, reduction in the expressions of stemness genes was observed in Rex-1 siRNA treated-tumor.

<6-4-3> Expressions of neural stem cell markers

Expressions of neural stem cell markers in the tumor tissues of Example <6-2> were investigated using Rex-1, Nestin and GFAP, the neural stem cell markers, primary antibodies by the same manner as described in Example <1-3>.

As a result, as shown in Figure 21, induction of GFAP and drastic reduction of Rex1 and Nestin were observed.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

The present invention to provide a pharmaceutical composition for treating BCNU(1,3-Bis(2-chloroethyl)-1-nitrosourea, Carmustine)-resistant glioblastoma multiforme. The pharmaceutical composition of the present invention can be effectively used for the treatment of BCNU-resistant glioblastoma multiforme by co-administration with BCNU.

Claims

A pharmaceutical composition comprising Rex-1 target sequence specific siRNA as an active ingredient for treating BCNU(1,3-Bis(2-chloroethyl)-1-nitrosourea, Carmustine)-resistant glioblastoma multiforme.
The pharmaceutical composition according to claim 1, wherein the Rex-1 target sequence is characteristically the sequence represented by SEQ. ID. NO: 17 or NO: 18.
The pharmaceutical composition according to claim 1, wherein the siRNA contains an independent sense RNA strand homologous to the said target sequence and an antisense RNA strand complementary thereto or is single RNA strand having stem-loop structure where the said sense RNA strand and the antisense RNA strand are linked by loop.
The pharmaceutical composition according to claim 3, wherein the sense RNA strand is characteristically the sequence represented by SEQ. ID. NO: 17 or NO: 18.
A pharmaceutical composition comprising Rex-1 target sequence specific siRNA and BCNU as active ingredients for treating BCNU-resistant glioblastoma multiforme.
The pharmaceutical composition according to claim 5, wherein the Rex-1 target sequence is characteristically the sequence represented by SEQ. ID. NO: 17 or NO: 1
The pharmaceutical composition according to claim 5, wherein the siRNA contains an independent sense RNA strand homologous to the said target sequence and an antisense RNA strand complementary thereto or is single RNA strand having stem-loop structure where the said sense RNA strand and the antisense RNA strand are linked by loop.
The pharmaceutical composition according to claim 7, wherein the sense RNA strand is characteristically the sequence represented by SEQ. ID. NO: 17 or NO: 18.
A method for treating BCNU-resistant glioblastoma multiforme containing the step of administering a pharmaceutically effective dose of Rex-1 specific siRNA to a subject with BCNU-resistant glioblastoma multiforme.
The method according to claim 9, wherein the Rex-1 target sequence is characteristically the sequence represented by SEQ. ID. NO: 17 or NO: 18.
The method according to claim 9, wherein the siRNA contains an independent sense RNA strand homologous to the said target sequence and an antisense RNA strand complementary thereto or is single RNA strand having stem-loop structure where the said sense RNA strand and the antisense RNA strand are linked by loop.
The method according to claim 11, wherein the sense RNA strand is characteristically the sequence represented by SEQ. ID. NO: 17 or NO: 18.
The method according to claim 9, wherein the BCNU-resistant glioblastoma multiforme cells are isolated or cultured cells expressing Rex-1 gene.
A method for treating BCNU-resistant glioblastoma multiforme containing the step of co-administering Rex-1 target sequence specific siRNA and BCNU to a subject with BCNU-resistant glioblastoma multiforme.
The method according to claim 14, wherein the siRNA is treated a couple of hours before BCNU treatment for the co-administration.
A method for treating BCNU-resistant glioblastoma multiforme containing the step of co-administering Rex-1 target sequence specific siRNA, BCNU and Erk1/2 inhibitor to a subject with BCNU-resistant glioblastoma multiforme.
The method according to claim 16, wherein the Erk1/2 inhibitor is an Erk1/2 specific inhibitor or an Erk1/2 broad range inhibitor.
The method according to claim 17, wherein the Erk1/2 specific inhibitor is PD98059.
The method according to claim 17, wherein the Erk1/2 broad range inhibitor is AG1478.
A use of Rex-1 target sequence specific siRNA for the prevention and treatment of BCNU(1,3-Bis(2-chloroethyl)-1-nitrosourea, Carmustine)-resistant glioblastoma multiforme.