KR20110139541A - Radiation sensitizing composition for cancer theraphy comprising fts expression inhibiting agent as active ingredient and composition for protecting cell from radiation comprising fts protein or its expression construct as active ingredient - Google Patents

Abstract

PURPOSE: A composition containing FTS expression inhibitor for cancer cell radiation sensitizing is provided to induce cancer cell apoptosis and to improve radiation sensitivity of cancer cells. CONSTITUTION: A composition for cancer cell radiation sensitizing contains FTS expression inhibitor as an active ingredient. The inhibitor is siRNA or shRNA for a nucleotide sequence encoding FTS. The nucleotide sequence encoding FTS contains a sequence of sequence number 2 or 3. The shRNA contains a sequence of sequence number 4, 5, or 6. The composition contains pharmaceutically effective amount of the inhibitor and a pharmaceutically acceptable carrier. A composition for cell protection from radiation contains FTS protein or an expression construct of the nucleotide encoding FTS protein as an active ingredient.

Description

Radiation Sensitizing Composition For Cancer Theraphy Comprising FTS Expression Inhibiting Agent of Cancer Cells Comprising FTS Expression Inhibitors as Active Ingredients and Radiation Sensitizing Composition For Cancer Theraphy Comprising FTS Expression Inhibiting Agents As Active Ingredient and Composition For Protecting Cell From Radiation Comprising FTS Protein or Its Expression Construct As Active Ingredient}

The present invention protects the cells from radiation-sensitive composition of cancer cells comprising the expression inhibitor of FTS (Fused Toes Homologue) as an active ingredient and radiation comprising the FTS protein or expression-construct thereof It relates to a composition for use.

Although concurrent chemotherapy has demonstrated a remarkable improvement in the control and survival of pelvic disease, radiation resistance of tumor cells has become a major problem in treatment. Accordingly, new therapeutic targets or approaches are needed to address the radiation resistance problem. DNA damaging agents, such as ionizing radiation, inhibit the synthesis of DNA in cells with DNA damage in order to activate rapid biochemical reaction pathways to maintain genome robustness. The DNA damage response is a fundamental process because mutations in the pathway result in increased sensitivity to DNA replication errors, impaired immune function and increased risk of cancer development [6-9]. There are two main cell cycle targets of DNA damage response, G1 / S and G2 / M checkpoints. Damaged DNA runs a genetic program that causes the cell to stay at either of the two major checkpoints until the damage is repaired or the cell enters apoptosis. Although this response is complex, the best characterized elements of the DNA damage response include Ataxia Telangiectasia Mutated (ATM) serine / threonine kinases, p53 protein stabilization and terminal activation of transcriptional activation of p53-dependent genes [10]. Clear cell cycle related proteins are responsible for G1 / S and G2 / M arrest. In particular, p21 (Waf-1), a p53 target gene induced by ionizing radiation (IR), binds to the CDK4 / cyclinD and CDK6 / cyclinD complexes and inhibits the G1 / S transition. Targeted mutations in the p21 gene lead to late development of cancer in mice and loss of G1 arrest in cells exposed to DNA damaging agents [11-14]. Although the DNA damage response pathway is very important in maintaining genotoxic stress, the reaction product is very dependent on the cellular context. Clearly, other signaling pathways activated intracellularly during DNA damage can modulate the response and alter the product of DNA damage-induced signal transduction. Multiple mutations in tumor cells result in IR-induced cell cycle arrest and resistance to apoptosis, reducing the effect of radiation therapy [6-9]. Similarly, abnormal activation of survival, growth, and proliferation signaling pathways is interfered by DNA damage responses, allowing many of these pathways to proliferate despite the presence of DNA damage [15-18].

Uterine cervical cancer is the second most common cancer in the world for women despite the very effective screening of this cancer [1]. Radiation therapy can be used to treat all stages of cervical cancer. Radiation therapy, including adjuvant radiation therapy after surgical operation, is used in more than 60% of all cervical cancer treatments [2]. The most prominent change in standard radiotherapy of cervical cancer was cisplatin-containing concurrent chemotherapy with radiation therapy for patients with locally advanced stage disease [3-5].

The FTS (Fused Toes Homologue) gene was first identified as one of six genes missing in mouse variants called toe junctions due to defects in the development of limbs and was named FT1 / FTS. The mutation in the Ft mouse is located on the mouse chromosome 8, and it is estimated that several hundred kb of dye is missing, suggesting that several genes may be involved in the Ft phenotype [19]. To date, no evidence of direct association between the phenotype of Ft mice and the FTS gene has been reported. Homologous genes 96% homologous to FTS in mice have been identified in humans and have been suggested to interact with Akt1 to regulate the phosphorylation of Akt by PDK1 [8], but at present the intrinsic function of FTS Very little is known about it [20]. However, although the NH ₂ -terminal region of FTS exhibits extensive sequence homology with the Ubc domain conjugating enzyme, the lack of cysteine residues in the active site essential for enzymatic activity suggests that ubiquitin-mediated proteolysis It can be estimated that FTS is involved in the regulation [19]. Recent studies have reported that FTS specifically recognizes mono-ubiquitinated proteins to mediate transport in endosomes.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have studied in depth the mechanism of action on the radiation therapy resistance of cancer. As a result, when cancer cells are exposed to radiation, overexpression of the FTS (Fused Toes Homologue) gene is induced, and the expressed FTS protein is localized in the nucleus, and when the cancer cells are irradiated with FTS expression, It has been demonstrated that proliferation inhibition and killing of cancer cells is increased to a significant extent. In other words, the inventors of the present invention are proteins that impart resistance to radiation in cancer cells and normal cells, and inhibiting their expression can greatly improve the radiotherapy sensitivity of cancer cells and induce the expression of FTS proteins in normal cells. The present invention was completed by elucidating that cells can be protected from cell damage or cell death caused by radiation.

Accordingly, an object of the present invention is to provide a radiation-sensitive composition of cancer cells comprising the expression inhibitor of FTS (Fused Toes Homologue) as an active ingredient.

Another object of the present invention is to provide a composition for protecting cells from radiation comprising an FTS (Fused Toes Homologue) protein or an expression-construct of FTS protein as an active ingredient.

The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the present invention provides a radiation-sensitive composition of cancer cells comprising the expression inhibitor of FTS (Fused Toes Homologue) as an active ingredient.

According to a preferred embodiment of the present invention, the expression inhibitor of the FTS is siRNA or shRNA for the nucleotide sequence encoding the FTS.

According to another preferred embodiment of the present invention, the nucleotide sequence encoding the FTS comprises SEQ ID NO: 2 or 3 sequence.

According to another preferred embodiment of the present invention, the shRNA comprises SEQ ID NO: 4, 5, or 6 sequence.

According to another preferred embodiment of the present invention, the cancer is lung cancer, bone cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, breast cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, small intestine cancer, thyroid cancer, parathyroid cancer Prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney cancer or brain cancer.

According to another preferred embodiment of the present invention, the composition comprises (a) a pharmaceutically effective amount of an expression inhibitor of FTS (Fused Toes Homologue); And (b) a pharmaceutically acceptable carrier.

The present invention relates to a composition for inhibiting the expression of the FTS gene having radiation resistance activity of cancer cells to improve the radiation sensitivity of cancer cells during cancer radiation treatment.

The present invention is based on the fact that when cancer cells are exposed to radiation, the FTS gene is overexpressed and moved to the nucleus to localize to protect cancer cells from radiation, and inhibiting the expression of these FTS genes can greatly improve the radiation sensitivity of cancer cells. will be.

In the present invention, "FTS (Fused Toes Homologue) protein" refers to a protein also named Aktip (AKT interacting protein), preferably a protein having an amino acid sequence of SEQ ID NO: 1.

In the present invention, "FTS gene" refers to a gene including a nucleotide sequence encoding the FTS protein, more preferably has a nucleotide sequence of SEQ ID NO: 2 or 3 sequence.

As used herein, the term "radiation sensitizing" refers to a treatment for improving the sensitivity of radiation to cancer cells to increase the effectiveness of radiation therapy during radiation therapy of cancer. In other words, it means that the cancer cells are resistant to radiation, thereby inhibiting the cancer cells from inducing death.

The term "expression of FTS" in the present invention means that the nucleotide sequence encoding the FTS is transcribed and translated to produce the FTS protein. Thus, the expression "inhibit expression of FTS" in the present invention means that the production of FTS protein is inhibited.

In order to inhibit the expression of FTS in the present invention, for example, but not limited to RNA interference (RNA interference, RNAi) method can be used. RNAi can be induced by designing and synthesizing small interfering RNAs for the FTS gene and introducing them into cells.

As used herein, the term "RNAi" refers to a phenomenon in which double-strand RNA (dsRNA) specifically and selectively binds to a target gene and efficiently inhibits its expression by cleaving the target gene. For example, when dsRNA is introduced into a cell, expression of a gene having a homologous sequence to the introduced RNA sequence is suppressed (knockdown).

As used herein, the term "siRNA" refers to a nucleic acid molecule capable of mediating RNAi (WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 and WO 00/44914). .

The siRNA molecule of the present invention may have a structure in which a sense strand (a sequence corresponding to the FTS mRNA sequence) and an antisense strand (a sequence complementary to the FTS mRNA sequence) are positioned opposite to each other to form a double strand.

In addition, the siRNA molecules of the present invention may have a single chain structure with self-complementary sense and antisense strands. siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. May be included. The total length is 10-100 bases, preferably 15-80 bases, more preferably 20-70 bases. The siRNA terminal structure can be either blunt or cohesive ends. The cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures.

Design of siRNA in the present invention can be carried out as follows.

(Iii) It will not specifically limit, if it is a nucleotide sequence which codes FTS, Any arbitrary region of this sequence can be made into a candidate. For example, any region of the nucleotide sequence of the sequence listing second sequence or the third sequence, which is an FTS coding sequence, may be a candidate. That is, the siRNA for inhibiting FTS gene expression in the present invention has a sequence complementary to some region of the nucleotide sequence described in SEQ ID NO: 2 or 3 sequence. (Ii) Select a sequence starting with base AA in the selected region, wherein the sequence is 19-25 bases, preferably 19-21 bases. The GC content of this sequence is preferably selected to be 40-60%, for example. In order to introduce the prepared siRNA into the cell, a siRNA synthesized in vitro can be inserted into the plasmid DNA and introduced into the cell, and a method of annealing two RNAs can be employed.

In addition, the present invention may use shRNA (short hairpin RNA) to obtain the RNAi effect.

In the present invention, "shRNA" refers to an RNA molecule having a stem and loop structure in which some regions of one chain form a complementary chain with another region. shRNAs can be designed so that some of them form a stem and loop structure. For example, if the sequence of a region is sequence A and the complementary chain to sequence A is sequence B, these sequences are present in one RNA chain such that sequence A-spacer-sequence B is in order. Designed to be a total length of 45-60 bases. For example, sequence A is a sequence of a portion of the target FTS gene (SEQ ID NO: 2 or 3), and the target region is not particularly limited, and may be any region. And the length of sequence A is 19-25 bases, preferably 19-21 bases.

After synthesizing the shRNA base sequence, it is cloned into a plasmid vector or a viral vector (lentiviral or adenovirus) to prepare an expression construct, which is introduced into the cell and expressed in the cell. Is formed. shRNA is converted to siRNA by Dicer in the cell to show RNAi effect. Expression constructs or vectors comprising shRNAs can be prepared by methods known in the art (see US Patent Publications 20040106567 and 20040086884).

In the present invention, "cancer", which is the target of radiation therapy, is not limited to a specific type of cancer, for example, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer , Ovarian cancer, rectal cancer, stomach cancer, anal muscle cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer , Parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) ) Tumors, primary central nervous system lymphoma, spinal cord tumors, brain stem glioma, pituitary adenoma, and the like. According to the most preferred embodiment of the invention said cancer is cervical cancer.

The composition of the present invention comprises (a) a pharmaceutically effective amount of an expression inhibitor of FTS (Fused Toes Homologue); And (b) it can be prepared in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers of the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. no.

In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, or the like. Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-100 mg / kg body weight.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, the present invention is directed to a radiation comprising an (i) Fused Toes Homologue (FTS) protein or (ii) an expression-construct of an FTS protein encoding polynucleotide sequence as an active ingredient. Provided are compositions for use in protecting cells.

The inventors have experimentally demonstrated that irradiation results in increased expression of the FTS (Fused Toes Homologue) protein in cells and that the protein functions to protect cells from cell damage or cell death by radiation. Thus, the present invention offers the possibility of protecting the cells from radiation induced cell damage and apoptosis by introducing or increasing the expression of FTS protein in normal cells.

According to a preferred embodiment of the present invention, the FTS protein has the amino acid sequence of SEQ ID NO: 1.

According to another preferred embodiment of the present invention, the FTS protein coding polynucleotide sequence has SEQ ID NO: 2 or 3 sequence.

According to another preferred embodiment of the invention, the composition comprises (i) a pharmaceutically effective amount of an expression construct of (i) Fused Toes Homologue (FTS) protein or (ii) FTS protein encoding polynucleotide sequence; And (iii) a pharmaceutically acceptable carrier.

As used herein, the term "expression construct" means a minimum of elements for expression, including a polynucleotide sequence for expression purposes and an expression sequence (eg, a promoter) that directs the expression of the sequence.

Such expression constructs preferably comprise nucleotide sequence-polyadenylation sequences for transcriptional regulatory sequence-expression purposes.

The expression construct comprises, for example, a promoter-FTS-(Fused Toes Homologue) protein coding nucleotide sequence-poly adenylation sequence that is operable in eukaryotic cells.

As used herein, the term “promoter operable in eukaryotic cells” means a transcriptional regulatory sequence capable of inducing transcription of a gene of interest in eukaryotic cells.

In the present invention, the FTS protein coding nucleotide sequence is operably linked to an operable promoter.

As used herein, the term “operably linked” means a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, thereby The regulatory sequence will control the transcription and / or translation of said other nucleic acid sequence.

The promoter bound to the FTS protein coding nucleotide sequence is preferably capable of controlling transcription of the subunit sequence by operating in animal cells, more preferably mammalian cells, and promoters and mammalian cells derived from mammalian viruses. Promoters derived from the genome of, for example, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, EF1 alpha promoter, metallothionine Promoters, beta-actin promoters, promoters of human IL-2 genes, promoters of human IFN genes, promoters of human IL-4 genes, promoters of human lymphotoxin genes, and promoters of human GM-CSF genes It is not.

In the present invention, the poly-aninylation sequence bound to the FTS protein coding nucleotide sequence is a plasmonic hormone terminator (Gimmi, ER, et al., Nucleic Acids Res. 17: 6983-6998 (1989)), SV40-derived poly Adenylation sequence (Schek, N, et al., Mol. Cell Biol. 12: 5386-5393 (1992)), HIV-1 polyA (Klasens, BIF, et al., Nucleic Acids Res. 26: 1870-1876 ( 1998)), β-globin polyA (Gil, A., et al, Cell 49: 399-406 (1987)), HSV TK polyA (Cole, CN and TP Stacy, Mol. Cell. Biol. 5: 2104-2113 (1985)) or polyomavirus polyA (Batt, D. B and GG Carmichael, Mol. Cell. Biol. 15: 4783-4790 (1995)).

Description of the pharmaceutical composition comprising an expression-construct of a FTS (Fused Toes Homologue) protein or an FTS protein encoding polynucleotide sequence is directed to a radiosensitivity of cancer cells comprising the expression inhibitor of FTS as an active ingredient. The description is the same as that for the form of the pharmaceutical composition in the acting composition, and therefore are not redundantly described in order to avoid excessive complexity herein.

The present invention relates to a radiation-sensitive composition of cancer cells comprising the expression inhibitor of FTS (Fused Toes Homologue) as an active ingredient. The present invention also relates to a composition for use in protecting cells from radiation comprising an FTS protein or an expression construct of FTS protein as an active ingredient. The composition of the present invention inhibits the expression of FTS having cytoprotective ability against radiation, thereby increasing the radiation sensitivity of cancer cells when irradiating the radiation, inducing apoptosis, stopping at G0 / G1 stage of the cell cycle, and the like. Greatly improves sensitivity to Therefore, the composition of the present invention can greatly reduce the radiation resistance of cancer cells, thereby improving the treatment efficiency during radiation chemotherapy. In addition, the cell protective composition of the present invention has the effect of protecting the cells from cell damage or cell death induced by irradiation.

1A shows the tissue staining results of FTS. Panels (a) to (e) show the results of FTS staining from DFS patient samples, and all DFS cancer samples show cytoplasmic localization of FTS. Panels (f)-(h) are photographs of recurrent tissue and show that FTS immunostaining shows that FTS is localized in the nucleus (Bar = 50 μm).
1B shows that endogenous FTS localizes into the nucleus after irradiation in HeLa cells.
1C shows that exogenous Myc-FTS localizes into the nucleus after irradiation in HeLa cells.
2A shows differential expression of FTS in cervical cancer cells. Irradiation in all four types of cervical cancer cells induced overexpression of FTS, while HeLa cells showed the highest expression change, while caSki cells showed the lowest expression change level.
2B is a result demonstrating the effect of shRNA and down regulation of activated Akt after irradiation in FTS knockdown HeLa cells.
2C shows PARP cleavage related only to knock-down and irradiation of FTS.
2D shows that the irradiated HeLa-siFTS cells had a 4-fold increase in caspase-3 over a defined time interval.
FIG. 3A shows the reduction in colonogenic survival of HeLa cells after knockdown of FTS expression by shRNA transfection.
3B shows that transient overexpression of Myc-FTS in HEK293 cells shows a higher survival fraction compared to untransfected HEK293 cells.
4A shows the results of an increase in apoptosis in FTS knock-down HeLa-siFTS cells compared to HeLa-siV after irradiation as a result of Annexin V staining and flow cytometry. Y axis represents PI labeled population and X axis represents FITC-labeled Annexin V positive cells.
4B shows that the histogram compares the levels of cells causing atoptoses between HeLa-siV and HeLa-siFTS cells in a time dependent manner.
5A shows the change in cell cycle distribution after FTS knock-down in response to irradiation. Puromycin selected shRNA transfected cells were treated with a single radiation dose of 4 Gy and incubated continuously for 48 hours. Cell cycle distribution in each sample was determined by flow cytometry.
5B shows that G1 arrest is resolved in a concentration dependent manner by transient Myc-FTS expression after 4Gy irradiation in HEK293 cells.
FIG. 5C shows the effect of inhibition of FTS expression in response to irradiation by analyzing protein expression of early G1 cyclins D1, D3, CDK's CDK4, 6 and their inhibitors p21, p27, p16 and p15 Shows the result of measurement. The expression of cyclin D1 and D3 was dramatically reduced by FTS knockdown (Panels 2 and 3). The expression of key modulators of CDK6 and early G1 decreased with or without a slight change in CDK4 (Panels 4 and 5). Importantly, expression of p21 and p27 was downregulated by FTS knockdown, with a slight increase in other CDK inhibitors of p16 but no change was observed in p15 (Panels 8 and 9). Expression of β-actin was measured as a control.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Materials and Methods

1. Patient Sample

Formalin-fixed paraffin-sealed samples of cervical cancer tissue of patients undergoing curative radiation therapy from May 2003 to July 2005 at Chungbuk National University Hospital were used. All experiments were conducted with the approval of the Institutional Review Board (IRB) of Chungbuk National University Hospital.

2. Cell Culture and Transfection

HeLa, ME180, SiHa and CaSki cell lines were obtained from KCLB / KCLRF Korea Cell Line Bank (Seoul, Korea). In all experiments, cells were treated with RPMI 1640 or DMEM medium supplemented with 10% (v / v) fetal calf serum (FCS) (Serum Source International, Charlotte, USA), 100 U / ml penicillin and 100 mg / ml streptomycin. WelGENE, Daegu, Korea) were used to incubate in a humid incubator at 37 ° C. and 5% CO ₂ . ShRNA plasmid DNA transfection of HeLa cells was performed using Enhancer-QTM Reagent and WelFect-ExTM plus Reagent (WelGENE, Daegu, Korea) according to the instructions provided by the manufacturer. Comparative expression of the transfected construct at 24 hours after transfection was confirmed by Western blotting.

3. Irradiation

Irradiation was done when the cells reached 80% subconfluency. Cells were examined 24 hours after transfection in a transfection experiment. Cells were examined using a 6 MV linear accelerator (Siemens Mevatron M6700, Concord, CA, USA) at a 3 Gy / min dose. The cells were then incubated in the growth chamber for a specific time (1-48 hours at 37 ° C.) prior to each assay.

4. FTS shRNA Constructs and Stable Knockdown

FTS knockdown was done by RNA interference (RNAi) using vectors based on the shRNA approach: 5'-CCUUGGUUGUGAAGCAGAA-3 '(SEQ ID NO: 4); 5'-GUUCGAUAUUCCUGUCUUU-3 '(SEQ ID NO: 5); Cloned shRNA constructs targeting 3 different portions of the FTS protein, encoding 5′-GUCUUAGGGUGGUUAUCUA-3 ′ (SEQ ID NO: 6), were purchased from Santacruz Biotechnology (California, USA). Stably down-regulated clones were selected using 3 μg / ml dmf of puromycin in RPMI1640 medium.

5. Antibodies and Reagents

Antibodies against FTS protein (ab56407) were purchased from Abcam (Cambridge, UK) and anti-p-AKT, anti-p21, p27, p16, p15, CDK4, CDK6, Cyclin D1 and D3 antibodies were described in Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin, anti-AKT, anti-PARP, anti-Bcl2, anti-Bax antibody and secondary antibody anti-mouse, anti-goat, anti-rabbit are Santa Cruz Biotechnology (Santa Cruz, CA) , USA). Propidium iodide, RNase A, dithiothreitol (DTT), Aprotinin, and phenylmethylsulfonyl fluoride were purchased from Sigma (St. Louis, MO, USA). Polyvinylidene difluoride membrane was purchased from Bio-Rad (CA, USA).

6. Immunofluorescence

HeLa, ME180, SiHa and CaSki cell lines were incubated on a cover slip until reaching 70% confluence and fixed with 4% paraformaldehyde. After brief washing, the cells were blocked with 10% FBS in PBST and the blocked cells were treated for 1 hour using anti-FTS primary antibody. Expression was measured using Alexa-488 conjugated Gout anti-mouse secondary antibody and Confocal microscope Leica DM IRB (Germany).

7. Immunohistochemistry

Immunohistochemical staining was performed on paraffin-sealed tissue sections using the Immunohistochemistry Accessory Kit (Bethyl Laboratories Montgomery, TX, USA) following recovery of antigen. Briefly, the paraffin-free section was incubated for 6 minutes in a microwave oven using citrate buffer pH 6.0 to recover antigen. The endogenous peroxidase was blocked for 20 minutes with 0.6% hydrogen peroxide in methanol. Tissue sections were treated for 30 minutes with protein blocking solution. Primary antibody (dilution FTS 1: 100, p21 1:50) was added to the slides and incubated overnight at 4 ° C. After washing in PBST, sections were incubated with suitable secondary antibodies for 60 minutes. Color reactions were developed using Liquid DAB substrate Pack (Bethyl Laboratory, Montgomery, TX, USA). Sections were counterstained with hematoxylin.

8. Gel Electrophoresis and Immunoblotting

The cells were harvested and pelleted in 400 μl of lysis buffer [20 mM Hepes (pH 7.4), 2 mM EGTA, 50 mM glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mM PMSF, 10 μg / ml leupetin ( leupeptin), 10 μg / ml aprotinin and 10 μg / ml pepstatin] and suspended on ice for 30 minutes. Laemmli's sample buffer [0.625 M Tris ?? HCl (pH 6.8), 10% β-mercaptoethanol, 20% SDS, 20% glycerol, 0.004% bromophenol blue) after centrifugation at 15,000 rpm for 15 minutes at 4 ° C. Three times the volume was added to the supernatant and boiled for 5 minutes. The protein was separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Bio-Rad, CA, USA). The membranes were incubated overnight at 4 ° C. with each antibody in TBST buffer containing 10% skim milk (10 mM Tris ?? HCl, 0.1 M NaCl, 0.1% Tween 20, pH 7.4). Each antibody was detected with Intron, WestSol ™, Chemiluminescence Reagent Plus. Using HRP-conjugated anti-rabbit, anti-mouse, or anti-Gout IgG antibodies.

9. Cell Growth Curve

Wild type normal and stabilizing cells were trypsinized and seeded at a rate of 1 × 10 ³ per well in 6 well plates. Cells were then counted for each day after trypsinization and plating. Counted cell numbers were recorded to draw growth curves. The growth curve was repeated three times, two times each.

10. Colonic cell survivial assay

Survival analysis was performed according to known methods [21]. Briefly, cells are irradiated, trypsinized, counted with a hemocytometer, plated at clone density in 60-mm tissue culture plates, and once a week for two weeks. Cultures were incubated with exchange feed. The inoculation density allowed a constant 1000 cells per 60 mm dish. On day 14 of culture cells were fixed in methanol: acetic acid = 7: 1 and stained with 0.5% crystal violet. Colonies were counted if they contained more than 50 cells. Colony-forming efficiency was calculated by dividing the number of colonies by the number of plated cells and comparing with the control plate. The viable portion of the treated plate was calculated as the colonization efficiency of the plate from the treated cells divided by the colonization efficiency of the control cells. Survival curves were performed by at least three replicate experiments.

11. Cell cycle analysis

HeLa cells were harvested by 0.25% (v / v) trypsinization and centrifuged at 1000 rpm for 5 minutes. Cells were fixed with 1: 1 PBS (13.7 mM NaCl, 0.27 mM KCl, 0.43 mM NaH ₂ PO ₄ 7H ₂ O, 0.14 mM KH ₂ PO ₄ (pH 7.5)) and ethanol (100%) solution. Cells were washed twice with PBS and incubated for 30 minutes at 37 ° C with 40 μg / ml propidium iodide (Sigma). Cells causing apoptosis were evaluated by flow cytometry using a FACScalibur ^™ cytometer (Becton-Dickinson, San Jose, Calif., USA). After cells were excited at 488 nm using a 15 mW argon laser, fluorescence was monitored by fluorescence channel FL2 yellow with a default filter at 585/42 nm. Ten thousand events were induced by forward side scatter (FSC) signals and gated electronically for FL2-W / FL2-A (W is width and A is area) to rule out cell clumps. The data was then analyzed using the ModiFit LT ^™ program.

9. Measurement of Apoptotic Cells by Flow Cytometry Using Annexin V

Exposure of phosphatidylserine (PS) was assessed by measuring annexin V-FITC binding. Briefly, about 4 × 10 ⁵ cells were harvested and washed twice in 1 ml cold PBS (pH 7.4). The cells were then resuspended in 200 μl binding buffer (pH 7.4) and 10 μl of annexin V-FITC and 5 μl of PI were added. After 15 min incubation at room temperature, 300 μl of binding buffer was added to each sample and analyzed immediately within 1 hour by flow cytometry (Becton-Dickinson, San Jose, Calif., USA).

10. Caspase-3 Activity Assay

The activity of caspase-3-like protease in cell lysates was measured using a colorimetric caspase-3 assay kit (Sigma) with minor modifications according to the manufacturer's protocol. Briefly, the reaction mixture (total volume, 1000 μl) was prepared in 100 μl of cell lysate and 10 μl of caspase-3 substrate, acetyl-Asp-Glu-Val-Asp-p-nitroanilide in buffer. Final concentration, 200 μM). To illustrate nonspecific hydrolysis of the substrate, control reaction mixtures were prepared in assay buffer with 100 μl of cell lysate, 10 μl of substrate and 10 μl (final concentration, 20 μM) of specific caspase-3 inhibitor, acetyl-DEVD -CHO was included. Both mixtures were incubated overnight at 37 ° C. and absorbance was measured at 405 nm using an Evolution 5000 UV Visible spectrophotometer (Thermo Electron Corporation, Madison, Wis., USA). Absorbance data obtained using caspase-3 inhibitors were corrected for nonspecific hydrolysis by subtracting from measurements without caspase-3 inhibitors. In consideration of the container control and the blank, caspase-3 protein was used as a positive control.

Experiment result

1. Expression of FTS in Tissues of Cervical Cancer Patients

To investigate the role of FTS in contributing to radiation resistance in human cervical cancer cells, cervical tissue with various pathological conditions, including 24 sample of DFS (free survival) patient and 10 samples of recurring radiation therapy, Expression of FTS was analyzed. Interestingly, moderate to strong FTS immunostaining was observed in all 24 DFS samples, and staining of FTS was mainly detected in the cytoplasm (Panels a to panel d of FIG. 1A). Only one sample of recurrent epithelium showed negative results for FTS and the remaining nine samples showed variable FTS levels with all samples showing in-nuclear localization of FTS (panels e to panel h in FIG. 1A). These observations suggest that there is a functional relationship between radiation therapy resistance and FTS expression in cervical cancer.

Table 1 below shows the results for immunohistochemistry and localization for the expression of FTS in DFS and recurrent samples.

2. by irradiation FTS Cancer cells Nuclear Localization

Based on the above results, the expression of FTS was also measured for four types of cervical cancer cell lines. The localization of FTS was examined by immunofluorescence after 4G irradiation. Immunofluorescence data confirmed that the FTS protein was localized in the nucleus in cervical cancer cell lines ME180, SiHa and CaSki cell lines. In contrast, only HeLa cell lines showed nuclear / cytoplasmic localization (FIG. 1B). Intranuclear localization of FTS induced by radiation was further confirmed by immunofluorescence with Myc FTS transiently overexpressed in HeLa cells (FIG. 1C). Overexpressed Myc-FTS was completely transferred into the nucleus after 4G irradiation in a time dependent manner. The localization data and data in patient samples suggest that FTS plays a functional role in radiation therapy resistance in cervical cancer.

3. Induction of Increased Expression of FTS by Irradiation

To find a suitable in vitro cell line model to explain the role of FTS in radiation resistance, time-dependent FTS expression levels were exposed after exposure of 4 Gy radiation doses to four types of cervical cancer cell lines, HeLa, ME180, CaSki, and SiHa. Analysis was by Western blotting in a phosphorous manner. All cells showed high expression levels except CaSki, which showed very low expression under normal conditions. Interestingly all four cells showed upregulation of FTS protein expression after irradiation (FIG. 2A). HeLa cells were selected for further in vitro analysis.

4. Effect of radiation resistance on cancer cells by FTS

To determine the role of FTS for irradiation in cervical cancer cells, we established a stable FTS-silenced HeLa-siFTS through lentiviral mediated transfection and puromycin selection. It was. Cells containing the empty vector (HeLa-siV) were included as controls. Confirmation of decreased expression of FTS in HeLa-siFTS cells was confirmed at the protein level by western blotting compared to HeLa-siV cells (FIG. 2B).

Reduction of FTS protein in HeLa-siFTS cells was associated with a decrease in cell proliferation and growth. To assess the cytoprotective survival pathways related to the radioresistant properties of FTS mediated cervical cancer cells, the levels of phospho-AKT in HeLa-siV and HeLa-siFTS cells were analyzed before and after irradiation. In control cells, AKT phosphorylation was upregulated by irradiation, whereas irradiation did not induce AKT activation in HeLa-siFTS cells. Irradiation did not affect the level of total AKT (FIG. 2B). The experimental results are in agreement with previous findings that FTS will co-operate with AKT / PKB (20). The cytoprotective role of activated AKT has been studied very well in the radiation resistance of many cancers. Thus, the cooperation of the two molecules explains the radiation resistance capacity of cervical cancer cells.

5. Inhibition of FTS expression and induction of cell death by irradiation

To further assess whether FTS knockdown and PKB downregulation contribute to growth inhibition and killing of these cells, PARP cleavage levels and caspase-3 activity were analyzed. Little PARP cleavage was observed after a certain time interval in the irradiated HeLa-siV cells, whereas PARP cleavage was observed only in the irradiated HeLa-siFTS cells (FIG. 2C). Caspase-3 levels were also observed in a similar pattern. Irradiated HeLa-siFTS cells had a 4-fold increase in caspase-3 levels in a time dependent manner (FIG. 2D). No noticeable changes were observed in the mitochondrial pro-apoptotic protein and anti-apoptotic proteins BAX and Bcl-2.

6. Increasing cell radiation sensitivity by suppressing FTS expression

To determine if radiation susceptibility is increased by FTS knockdown, colony survival analysis was performed by increasing the dose of irradiation to 2, 4, 6, 8 Gy for both HeLa-siV and HeLa-siFTS cells. As shown in FIG. 3A, only the FTS knockdown itself reduced the viable fraction of HeLa cells to 0.457 ± 0.017.

Such reduction in the cell viability fraction by irradiation was more clearly manifested by levels of 0.246 ± 0.009, 0.128 ± 0.002, 0.057 ± 0.002 and 0.021 ± 0.001 with 2, 4, 6 and 8 Gy doses. For the same dose, HeLa-siV cells maintained viable fractions at 0.800 ± 0.03, 0.444 ± 0.029, 0.243 ± 0.007 and 0.125 ± 0.001.

To determine whether blocking expression of FTS contributes to this effect, a transient overexpression of Myc-FTS in HEK293 cells and irradiation of 2, 4, 6 and 8 Gy were performed to reverse clonogenic assay. ) Was performed. Surprisingly, ectopic expression of Myc-FTS increased the surviving fraction of control cells by 0.102 fractions and the increased surviving fractions were also observed for subsequent radiation doses (FIG. 3B). These data suggest that FTS provides radiation resistance to cells and that stable expression-blocking of FTS in HeLa cells increases radiation induced cell death.

In order to investigate the mechanism by which FTS knockdown increases radiation sensitivity and leads to cell death, we focused on apoptosis of HeLa-siFTS after irradiation via annexin-V staining using flow cytometry. HeLa-siFTS and HeLa-siV cells were irradiated and analyzed at 1, 6, 24 and 48 hour intervals, with the apoptosis of control non-irradiated cells and irradiated cells being 11.52%, 11.72% and 12, respectively. , 69%, 19.31% and 24.55%. The percentage of apoptosis in HeLa-siFTS cells was about 2-4 times higher than that of control cells, 29.36%, 45.06%, 47.91%, 49.15% and 59.29%, respectively (FIGS. 4A and 4B). The data suggest that radiation sensitization of HeLa-siFTS cells results from an increase in the cell population in the process of apoptosis.

7. Cell Cycle Suspension at G0 / G1 Stage by Inhibition of FTS Expression

HeLa-siFTS cells used in the experiments were used for cell cycle analysis. Inhibition of the expression of FTS in HeLa-siFTS cells significantly reduced the cell population in the S phase compared to that of HeLa-siV cells, and cells accumulated in the G0 / G1 phase. Proportion of cells at G0 / G1, S and G2 / M stages in non-irradiated HeLa-siFTS cells compared to 59.86%, 34.08% and 6.06% of unirradiated HeLa-siV cells Was 67.18%, 17.44% and 15.38% (FIG. 5A). Such distribution patterns at the cell cycle stage were made more apparent by irradiation. Interestingly, the accumulation of cells in the G0 phase after irradiation increased dramatically from 39% to 62% at 48 hours after irradiation in a time dependent manner.

The cell cycle data suggest that HeLa-siFTS cells stop at the G0 / G1 stage after irradiation, and accumulation at the G1 stage indicates that an increase in apoptosis is associated with inhibition of expression of FTS in HeLa cells ( 5a).

Ectopic expression of FTS shows that HEK293 cells are cleared from G1 arrest after irradiation (FIG. 5B). This is the result of reaffirming the role of FTS in cell cycle regulation.

To determine the mechanism of molecular level of FTS knockdown mediated cell cycle arrest and accumulation at G0 / G1, expression of proteins involved in regulating cell cycle progression and cell survival was checked.

One of the most important checkpoints in the cell cycle is the G1 phase, which is regulated by D-type cyclins, cyclin-dependent kinase (CDK) 4 and CDK 6. FTS inhibition dramatically altered the protein expression of early G1 phase D type cyclins and CDKs in HeLa-siFTS cells (FIG. 5C). HeLa-siFTS cells showed a marked decrease in the expression of cyclin D1 and D3 proteins after irradiation (Panels 2 and 3 in FIG. 5C). Consistent with cyclin data, protein expression of early G1 CDK, CDK6 and CDK4, which regulates G1 / S phase transition, was significantly reduced in irradiated HeLa-siFTS cells (Panels 4 and 5 in FIG. 5C). The results suggest that HeLa cells are stopped at the GO and G1 stages by FTS knockdown. CDKs are under the control of CDK inhibitors such as p16, p21 and p27, as their clinical significance in cervical cancer has been previously described (22). Expression of CDK inhibitors in FTS knockdown cells and control cells was assessed using western blotting after 4 Gy irradiation (FIG. 5C). In the case of FTS inhibition, the expression of p21, p27 and p16 was significantly increased after irradiation. Of the three inhibitors, p21 was significantly increased by FTS knockdown and irradiation, while the other inhibitors p15 did not change (FIG. 5C, Panels 6-9). In conclusion, in the case of FTS knockdown, after irradiation, the cells ceased cell cycle at the G0 / G1 stage with increasing p21 expression, and at the sub-G1 stage suggesting apoptosis after irradiation. Cell cycle accumulation increased significantly.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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<110> Chungbuk National University Industry Academic Cooperation Foundation <120> Radiation Sensitizing Composition For Cancer Theraphy Comprising          FTS Expression Inhibiting Agent As Active Ingredient and          Composition For Protecting Cell From Radiation Comprising FTS          Protein or Its Expression Construct As Active Ingredient <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 292 <212> PRT <213> Homo sapiens <400> 1 Met Asn Pro Phe Trp Ser Met Ser Thr Ser Ser Val Arg Lys Arg Ser   1 5 10 15 Glu Gly Glu Glu Lys Thr Leu Thr Gly Asp Val Lys Thr Ser Pro Pro              20 25 30 Arg Thr Ala Pro Lys Lys Gln Leu Pro Ser Ile Pro Lys Asn Ala Leu          35 40 45 Pro Ile Thr Lys Pro Thr Ser Pro Ala Pro Ala Ala Gln Ser Thr Asn      50 55 60 Gly Thr His Ala Ser Tyr Gly Pro Phe Tyr Leu Glu Tyr Ser Leu Leu  65 70 75 80 Ala Glu Phe Thr Leu Val Val Lys Gln Lys Leu Pro Gly Val Tyr Val                  85 90 95 Gln Pro Ser Tyr Arg Ser Ala Leu Met Trp Phe Gly Val Ile Phe Ile             100 105 110 Arg His Gly Leu Tyr Gln Asp Gly Val Phe Lys Phe Thr Val Tyr Ile         115 120 125 Pro Asp Asn Tyr Pro Asp Gly Asp Cys Pro Arg Leu Val Phe Asp Ile     130 135 140 Pro Val Phe His Pro Leu Val Asp Pro Thr Ser Gly Glu Leu Asp Val 145 150 155 160 Lys Arg Ala Phe Ala Lys Trp Arg Arg Asn His Asn His Ile Trp Gln                 165 170 175 Val Leu Met Tyr Ala Arg Arg Val Phe Tyr Lys Ile Asp Thr Ala Ser             180 185 190 Pro Leu Asn Pro Glu Ala Ala Val Leu Tyr Glu Lys Asp Ile Gln Leu         195 200 205 Phe Lys Ser Lys Val Val Asp Ser Val Lys Val Cys Thr Ala Arg Leu     210 215 220 Phe Asp Gln Pro Lys Ile Glu Asp Pro Tyr Ala Ile Ser Phe Ser Pro 225 230 235 240 Trp Asn Pro Ser Val His Asp Glu Ala Arg Glu Lys Met Leu Thr Gln                 245 250 255 Lys Lys Pro Glu Glu Gln His Asn Lys Ser Val His Val Ala Gly Leu             260 265 270 Ser Trp Val Lys Pro Gly Ser Val Gln Pro Phe Ser Lys Glu Glu Lys         275 280 285 Thr Val Ala Thr     290 <210> 2 <211> 2193 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gttaaggtgt 840 gcactgctcg tttgtttgac caacctaaaa tagaagaccc ctatgcaatt agcttttctc 900 catggaatcc ttctgtacat gatgaagcca gagaaaagat gctgactcag aaaaagcctg 960 aagaacagca caataaaagt gttcatgttg ctggcctgtc atgggtaaag cctggctcag 1020 tacagccttt cagtaaagaa gagaaaacag tggcgactta agagatggtg aatctggtgc 1080 accatgcact ttcctgctag actctggcct agttcaagct gaccaatggc agaggactgc 1140 ctgaagagta aaactgtgtg aacaatgact gactgccagt gttttccatg tatgcatagg 1200 ttctaacagc agggtttgga aacctgtctc taagtaatgc attacttctg tcagaagtgt 1260 cttagggtgg ttatctagtt cagtactcca aattattggg gaccttgagg cttaagtaag 1320 tatttttctg aatataatgc taaaggtaag ttgcattcat ttaaactaat agagcagaca 1380 gaattcagca ctacttaata gtttataaat cagtggtttc agttgtatat atgttaggaa 1440 atggagaggt atagagagag caggttccat agctcagcac ttttaagtgg aagatcattt 1500 gaatctcagt cttcagcctg cactgatttg tagcctgcac tgtcttactg atttacaaac 1560 tgaaatcact gagaaatgtc tttagttcag tgagaagaaa ccagaacact tgttcctagt 1620 gttgtgttgt tttttttaag caaattactt actgtatttt tatggcagga 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gaagacatta acaggggacg tgaaaaccag 300 tcctccacga actgcaccaa agaaacagct gccttctatt cccaaaaatg ctttgcccat 360 aactaagcct acatctcctg ccccagcagc acagtcaaca aatggcacgc atgcgtccta 420 tggacccttc tacctggaat actctcttct tgcagaattt accttggttg tgaagcagaa 480 gctaccaggc gtctatgtgc agccatctta tcgctctgca ttaatgtggt ttggagtaat 540 attcatacgg catggacttt accaagatgg cgtatttaag tttacagttt acatccctga 600 taactatcca gatggtgact gtccacgctt ggtgttcgat attcctgtct ttcacccgct 660 agttgatccc acctcaggtg agctggatgt gaagagagca tttgcaaaat ggaggcggaa 720 ccataatcat atttggcagg tattaatgta tgcaaggaga gttttctaca agattgatac 780 agcaagcccc ctgaacccag aggctgcagt actgtatgaa aaagatattc agctttttaa 840 aagtaaagtt gttgacagtg ttaaggtgtg cactgctcgt ttgtttgacc aacctaaaat 900 agaagacccc tatgcaatta gcttttctcc atggaatcct tctgtacatg atgaagccag 960 agaaaagatg ctgactcaga aaaagcctga agaacagcac aataaaagtg ttcatgttgc 1020 tggcctgtca tgggtaaagc ctggctcagt acagcctttc agtaaagaag agaaaacagt 1080 ggcgacttaa gagatggtga atctggtgca ccatgcactt tcctgctaga ctctggccta 1140 gttcaagctg accaatggca gaggactgcc tgaagagtaa aactgtgtga acaatgactg 1200 actgccagtg ttttccatgt atgcataggt tctaacagca gggtttggaa acctgtctct 1260 aagtaatgca ttacttctgt cagaagtgtc ttagggtggt tatctagttc agtactccaa 1320 attattgggg accttgaggc ttaagtaagt atttttctga atataatgct aaaggtaagt 1380 tgcattcatt taaactaata gagcagacag aattcagcac tacttaatag tttataaatc 1440 agtggtttca gttgtatata tgttaggaaa tggagaggta tagagagagc aggttccata 1500 gctcagcact tttaagtgga agatcatttg aatctcagtc ttcagcctgc actgatttgt 1560 agcctgcact gtcttactga tttacaaact gaaatcactg agaaatgtct ttagttcagt 1620 gagaagaaac cagaacactt gttcctagtg ttgtgttgtt ttttttaagc aaattactta 1680 ctgtattttt atggcaggag ggagaaaaag tgttacaacg gtttctaatg aagtccggta 1740 tttaaatgat aaatgactaa tgtgtttagt agagacaaaa taaaccaata aatgattgtt 1800 ctttgccatt tatgcaggaa actacccttt tctcaatata accaaacaaa ggctaattta 1860 taaatgcttt attgaaaaat acacttatct tcatataaaa ttacagtagc agtatcttga 1920 gaagttttat aaatattttt gcagaacact attctaattg aacaatgtaa gttccatatt 1980 tctctcagca atatgaagtt acctagtaac tttgtttata ctgattcaat ttacaattga 2040 attttctccc taataagatt attaatttga cttgaaaact gctggaacaa tagtgattaa 2100 taaagatatg tatagataat tcagctgtta aataacattt tctaatttgt ataaatggta 2160 ctatggtact aataaaaacc taaattctcc aaccaatttt tttaaactgc caggaacacc 2220 ca 2222 <210> 4 <211> 19 <212> RNA <213> Homo sapiens <400> 4 ccuugguugu gaagcagaa 19 <210> 5 <211> 19 <212> RNA <213> Homo sapiens <400> 5 guucgauauu ccugucuuu 19 <210> 6 <211> 19 <212> RNA <213> Homo sapiens <400> 6 gucuuagggu gguuaucua 19

Claims (10)

Radiation-sensitive composition of cancer cells comprising the expression inhibitor of FTS (Fused Toes Homologue) as an active ingredient.
The composition of claim 1, wherein the expression inhibitor of FTS is siRNA or shRNA for a nucleotide sequence encoding FTS.
3. The composition of claim 2, wherein the nucleotide sequence encoding the FTS comprises a second or third sequence.
The composition of claim 2, wherein the shRNA comprises SEQ ID NO: 4, 5, or 6.
According to claim 1, wherein the cancer is lung cancer, bone cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, breast cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, small intestine cancer, thyroid cancer, parathyroid cancer, prostate cancer, A composition characterized in that it is chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney cancer or brain cancer.
The composition of claim 1, wherein the composition comprises (a) a pharmaceutically effective amount of an expression inhibitor of FTS (Fused Toes Homologue); And (b) a pharmaceutically acceptable carrier.
A composition for cell protection from radiation, comprising (i) a FTS (Fused Toes Homologue) protein or (ii) an expression-construct of an FTS protein coding nucleotide sequence as an active ingredient.
8. The composition of claim 7, wherein the FTS protein has the amino acid sequence of SEQ ID NO: 1.
8. The composition of claim 7, wherein the FTS protein coding nucleotide sequence has a second or third sequence in SEQ ID NO.
8. The composition of claim 7, wherein the composition comprises a pharmaceutically effective amount of (i) a Fused Toes Homologue (FTS) protein or (ii) an expression-construct of an FTS protein coding nucleotide sequence; And (iii) a pharmaceutically acceptable carrier.

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