KR101371706B1 - Amniotic Fluid-derived Stem Cells Expressing Suicide Genes and Composition for Treatment of Cancer Comprising the Same - Google Patents

Abstract

The present invention provides a composition for treating cancer, comprising a mammalian amniotic fluid-derived stem cell expressing a suicide gene. More specifically, the present invention provides a pharmaceutically effective amount of amniotic fluid-derived stem cells and anticancer prodrugs transformed with an expression vector comprising a suicide gene; And it provides a pharmaceutical composition for treating cancer comprising a pharmaceutically acceptable carrier. The composition of the present invention has an excellent effect on cancer treatment by converting prodrugs into anticancer substances with cytotoxicity.

Description

Amniotic Fluid-derived Stem Cells Expressing Suicide Genes and Composition for Treatment of Cancer Comprising the Same}

The present invention relates to an amniotic fluid-derived stem cell expressing a suicide gene transformed with an expression vector comprising a suicide gene coding nucleic acid molecule and a pharmaceutical composition for treating cancer comprising the transformed amniotic fluid-derived stem cell as an active ingredient.

Cancer (cancer) refers to the mass of abnormally changed cells, incompletely mature and overproliferated, causing a variety of cancers worldwide cause social problems. Surgical resection, radiation, and anticancer cancer treatment methods, which are now known as cancer treatment techniques, have been greatly developed. These cancer treatment techniques are very useful but have limitations in that they are non-permanent treatments with the potential for recurrence and cause a variety of serious side effects. Anti-cancer agents commonly used in cancer treatment kill various normal cells including cancer cells, causing various side effects such as myelosuppression, vomiting, diarrhea, stomatitis, and hair loss (Hippokratia; 15: 93-95). These problems require new safer and more effective cancer treatment technologies. Accordingly, there is a recent interest in cell therapies. In particular, research on the characteristics and potential of stem cells is being actively conducted.

Stem cells are known as pluripotent cells that can multiply into various cells constituting the human body or are self-renewal. Stem cells can be roughly divided into embryonic stem cells, adult stem cells. Embryo stem cells are cultured very quickly and easily but contain various ethical problems and have a problem of inducing tumors when administered in vivo. Adult stem cells have no ethical problems than embryonic stem cells, but there are difficulties in culture and separation. Therefore, in the present invention, less ethical problems than embryonic stem cells, and when used in vivo, used amniotic fluid stem cells (human amniotic fluid-derived stem cells) that do not form tumors and are useful for culture and isolation. In addition, amniotic fluid-derived stem cells are inferior in their ability to differentiate into various cells and their self-renewal ability compared to embryonic stem cells. Amniotic fluid stem cells are isolated from the mother's amniotic fluid and can be obtained without relatively damaging the fetus and mother (Paolo De Coppi et al., Nat Biotechnol 25: 100-106 (2007).

Currently, technology for treating cancer using stem cells into which a suicide gene has been introduced uses embryonic stem cells or adult stem cells, and among them, mesenchymal stem cells are used as cell delivery systems. (Aboody K et al., Neuro) Oncol 8: 119-126 (2006), Tokuyama et al., Cancer Lett 291: 256-262 (2010). Suicide genes are genes that convert prodrugs that are harmless to the human body into cytotoxic anti-cancer substances. Suicide gene therapy using suicide genes / prodrugs is not only a cancer cell to which suicide genes are introduced, but also a suicide gene. The cancer cells around the unintroduced are also treated by the “bystander effect”, which is killed after the prodrug is administered. If the bystander effect can be enhanced, the gene therapy effect of suicide gene / prodrug can be maximized. have.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Republic of Korea Patent Application No. 10-2005-0091155 Republic of Korea Patent Application No. 10-2009-0062796

The present inventors earnestly researched to develop a cell therapy for cancer treatment using suicide genes. As a result, the suicide gene is introduced into amnion derived stem cells that have little ethical problems, do not form tumors when administered in vivo, and are easy to culture and isolate. When applied to cancer tissues confirmed the effect of greatly increasing cancer cell death has been completed the present invention.

Accordingly, it is an object of the present invention to provide amniotic fluid-derived stem cells expressing suicide genes.

Another object of the present invention is to provide a pharmaceutical composition for treating cancer comprising suicide gene-expressing amniotic fluid-derived stem cells as an active ingredient.

Still another object of the present invention is to provide an anticancer kit comprising a prodrug of amniotic fluid-derived stem cells expressing a suicide gene and an anticancer agent.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, the present invention provides a amniotic fluid-derived stem cell transformed with an expression vector comprising a nucleic acid molecule encoding a suicide gene.

As a result of earnest research efforts to develop a cell therapy for cancer treatment using suicide genes, the present inventors introduced suicide genes into amniotic fluid-derived stem cells that have little ethical problems, do not form tumors when administered in vivo, and are easy to culture and isolate. When applied to cancer tissues confirmed the effect of greatly increasing cancer cell death has been completed the present invention.

As used herein, the term “suicide gene” refers to a gene encoding a protein (eg, an enzyme) that does not have anticancer activity and therefore has the activity of converting a nontoxic prodrug into a cytotoxic anticancer drug. it means.

According to a preferred embodiment of the present invention, the suicide gene is a gene of cytosine deaminase (CD) and / or herpes simplex virus thymidine kinase (HSV-TK).

The cytosine deaminase (CD) is an enzyme that catalyzes a reaction to generate uracil by hydrolyzing an amino group using cytosine as a substrate. Cytosine deaminase produces 5-fluorouracil (5-FU) by deamino group reaction with 5-fluorocytosine (5-FC), a nontoxic prodrug. In addition, the produced 5-FU exhibits cancer cell specific toxicity of rapidly dividing cells by inhibiting the synthesis of DNA.

The herpes simplex virus thymidine kinase (HSV-TK) is an enzyme that catalyzes the reaction of introducing a phosphate group to its 5 'carbon position using a prodrug ganciclovir (GCV) as a reactant, and 5'-phosphorylated. Gancyclovir is converted to triphosphate-ganciclovir (GCV-TP), and GCV-TP exhibits cancer cell specific cytotoxicity by blocking DNA synthesis.

As used herein, the term "amniotic fluid-derived stem cells" refers to stem cells isolated from animal amniotic fluid.

Amniotic fluid-derived stem cells of the present invention are preferably stem cells isolated from mammalian amniotic fluid, more preferably human, swine, horse, cow, mouse, rat, hamster, rabbit, goat and sheep amniotic derived cells, Even more preferred are amniotic stem cells derived from humans, pigs, horses and cattle, most preferably human amniotic stem cells.

Since amniotic fluid stem cells are isolated from amniotic fluid, ethical problems are less likely to occur than embryonic stem cells, and when administered in vivo, they are less likely to form tumors. Very advantageous. In addition, amniotic fluid-derived stem cells are equivalent in their ability to differentiate into various cells and in self-renewal compared with embryonic stem cells.

In the present invention, the introduction of suicide genes into the amniotic fluid-derived stem cells is performed using a suitable expression vector, and for example, a plasmid vector or a viral vector can be used as the expression vector.

The viral vectors that can be used in the present invention are preferably retroviral vectors, baculovirus vectors, adenovirus vectors, Adeno-associated viruses (AVA) vectors, lentivirus vectors, herpes simplex virus vectors or bash Nia virus vectors, but are not limited thereto.

The origin of replication contained in the vector of the present invention includes, but is not limited to, f1 replication origin, SV40 replication origin, pMB1 replication origin, Adeno replication origin, AAV replication origin, and BBV replication origin.

Suicide genes in said expression vector are operably linked to a promoter. The term “operably linked” means a functional linkage between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulatory sequence To control transcription and / or translation of the nucleic acid sequence.

In the present invention, a promoter operably linked to a suicide gene to modulate the transcription of the suicide gene may be a promoter derived from the genome of a mammalian cell (eg, a metallothionine promoter) or a promoter derived from a mammalian virus (eg, Adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter and tk promoter of HSV) can be used.

Vectors of the present invention may comprise a polyadenylation sequence as a transcription termination sequence, for example, plastic field hormone terminators (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)).

The vector used in the present invention may include an antibiotic resistance gene commonly used in the art as an optional marker, for example, empicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin Resistance genes for neomycin, puromycin and tetracycline.

According to a preferred embodiment of the present invention, the introduction of the suicide gene into the amniotic fluid derived stem cells can be performed using a retroviral vector. Retroviruses are suitably selected as gene transfer vectors because they insert their genes into the host's genome, carry large amounts of foreign genetic material, and have a broad spectrum of cells that can be infected. The production of expression vectors using retroviruses is described in Nicolas and Rubinstein "Retroviral vectors," In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, 494-513 (1988). It is described in detail, which is incorporated herein by reference. In detail, in order to construct a retroviral vector, a suicide gene is inserted into the retroviral genome instead of the sequence of the retrovirus to prepare an expression vector of the non-replicating virus. The resulting non-replicating viral vector was transduced into the packaging cells, cultured the transduced packaging cells, and filtered to obtain a retroviral solution. Genes can be introduced. Subsequently, the amniotic fluid-derived stem cells into which the suicide gene is introduced may be selected using a selection marker included in the retroviral vector.

Amniotic fluid-derived stem cells in which suicide genes are introduced in the present invention express suicide genes, thereby inducing death of cancer cells by converting prodrugs that are administered while continuing to proliferate into substances having anticancer activity.

According to another aspect of the present invention, the present invention provides an anticancer pharmaceutical composition comprising an amniotic fluid-derived stem cell transformed with an expression vector comprising a nucleic acid molecule encoding a suicide gene as an active ingredient. do.

According to another aspect of the present invention, the present invention is transformed into an expression vector comprising a nucleic acid molecule encoding (i) a suicide gene, a positive-derived stem cell expressing the suicide gene and (ii) a prodrug of an anticancer agent It provides a kit for anticancer.

As demonstrated in one specific example of the present invention, the amniotic fluid-derived stem cells stably expressing suicide genes in the present invention have a killing effect on cancer cells by converting prodrugs of anticancer drugs into anti-cancer substances that exhibit potent cytotoxicity. Exert.

According to a preferred embodiment of the present invention, the amniotic fluid-derived stem cells are introduced into one or more suicide genes of cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK).

According to another preferred embodiment of the present invention, the anticancer prodrug is one or more of 5-fluorocytosine (5-FC) and gancyclovir (GCV).

According to another preferred embodiment of the present invention, the amniotic fluid-derived stem cells together with cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK), in this case anticancer prodrug 5-fluorine Administration of locitocin (5-FC) and gancyclovir (GCV) together shows a synergistic anticancer effect by multiple drugs.

Cancers that can be treated by applying the composition of the present invention are breast cancer, lung cancer, stomach cancer, liver cancer, hematologic cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer colon Cancer fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumors, urethral cancer, prostate cancer, bronchial cancer, bone marrow cancer and the like.

The pharmaceutical composition of the present invention may be prepared in the form of a suspension typically containing cells. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions which are ready for the preparation of solution or dispersion. In all cases, the pharmaceutical agent in the form of injection must be sterile and fluid enough to facilitate injection.

The pharmaceutical composition of the present invention may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" means that it does not cause an allergic reaction or a similar adverse reaction when administered to a human. Such carriers include certain solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and preparations in pharmaceutically active materials is well known in the art.

The carrier of the pharmaceutical composition may be, for example, a solvent or dispersion medium containing water, saline, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils . Fluidity can be maintained by the use of a coating agent such as lecithin. To prevent microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like may be included, and may include an appearance system such as sugar or sodium chloride. In addition, agents for delaying absorption in the composition for prolonging the absorption action upon administration in vivo, for example, aluminum monostearate and gelatin may be included. Sterile injectable solutions are prepared by mixing the required amount of the active compound in a suitable solvent with the various other ingredients mentioned above, as required, followed by filtered sterilization.

The pharmaceutical composition of the present invention may preferably be administered by parenteral, intraperitoneal, intradermal, intramuscular, intravenous routes, and more preferably by direct infusion into a site comprising cancer cells or cancer tissues. Can be.

The pharmaceutical compositions of this invention are administered in therapeutically effective amounts in a manner compatible with the formulation. The dosage can also be adjusted according to the condition or condition of the subject to be treated. For parenteral administration with aqueous injection solutions, the solution should be suitably buffered as needed, first making the liquid diluent isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intradermal and intraperitoneal administration. In this regard, the contents of carriers, formulations, and media that can be used in the present compositions are well known in the art (see Remington's Pharmaceutical Sciences & quot ;, 1995, 15th Edition).

The pharmaceutical compositions of the present invention are prepared in unit dose form by being formulated 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.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.0001-1000 mg / kg on an adult basis.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention relates to amniotic fluid-derived stem cells expressing suicide genes and an anticancer pharmaceutical composition comprising the same as an active ingredient.

(Ii) Injecting the suicide gene-expressing amniotic fluid-derived stem cells of the present invention by stably expressing the suicide gene around the cancer cells, thereby converting the administered prodrug into an anticancer toxic drug and thereby causing cancer cell specific cytotoxic effects. Exert.

(Iii) In the present invention, by using amniotic fluid-derived stem cells in stem cells as a suicide gene delivery medium, the possibility of tumor development during in vivo administration is significantly reduced and safe.

(Iii) The amniotic fluid-derived stem cells of the present invention have almost the same level of differentiation and self-renewal as compared with embryonic stem cells, and have the advantage of eliminating ethical problems caused when using embryonic stem cells.

1 is a schematic diagram of a vector for expression of suicide genes constructed by ligating CD-TK cDNA at the multicloning site of the retroviral vector pLPCX.
Figure 2 is a schematic diagram showing the principle of cancer treatment technology using the amniotic fluid-derived stem cells introduced suicide gene.
3 is a result of evaluating the effect of prodrugs on the viability of breast cancer cells in vitro. Panel A is a graph of the results of evaluating the effects of 5-FC and 5-FU on breast cancer cell viability, and panel B is a graph of the results of evaluating the effect of GCV on breast cancer cell viability.
Figure 4 is a result of evaluating the cell death of breast cancer cells according to the amniotic fluid-derived stem cells and prodrug treatment conditions introduced suicide gene.
5 is a result of transplanting the amniotic fluid-derived stem cells into which a suicide gene is introduced into a breast cancer animal model and injecting a prodrug, showing the volume and changes in the breast cancer of the animal model. The graphs in Panels A and B show tumor volume and body weight of the animal model during the formation of the tumors of the animal models without suicide-generating amniotic fluid-derived stem cells, respectively. Panels C and D show graphs of tumor volume and tumor weight measured after transplantation of amniotic fluid-derived stem cells into which suicide gene CD-TK was introduced after tumor size reached 300 mm ³ in animal models of breast cancer. to be.
6 is a fluorescence staining result of confirming the presence of amniotic fluid-derived stem cells into which a suicide gene has been introduced after collecting breast cancer tissues from an animal model of breast cancer.
Figure 7 is a result of comparing the breast tissue containing the mammary gland of each experimental group through hematoxylin (hematoxylin)-eosin (eosin) staining.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  1: Isolation and Culture of Amniotic Fluid Stem Cells

Amniotic stem cells were obtained by centrifuging 10 ml of amniotic fluid (1000 rpm, 8 minutes) and adding 2 ml of Chang's medium (Irvine scientific, Santa Anna, CA, USA) to the cells obtained, and then adding the suspension to a 35 mm culture dish. The cells were cultured at 37 ° C. in a 5% CO ₂ cell incubator at two-day intervals, and the cell group was checked for density by about 0.25% trypsin-EDTA when the density of the cell group was about 90%.

Example  2: Construction of Amniotic Fluid-derived Stem Cells Incorporated Suicide Gene

AF2 cells, an immortalized amniotic fluid derived stem cell line, were established by infecting a retroviral vector encoding a v-myc oncogene in amniotic fluid derived stem cells. Cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) genes were introduced into immortalized stem cells (AF2 cells) isolated from human amniotic fluid. Amniotic fluid stem cell AF2.CD-TK was constructed. More specifically, to prepare a CD-TK overexpressing human amniotic stem cell line (AF2.CD-TK), CD-TK cDNA (SEQ ID NO: 1) was first ligated to the multicloning site of the retroviral vector pLPCX ( See FIG. 1). The PT67 amphotropic packaging cell line was infected with a recombinant retroviral vector, the supernatant of the packaging cells was taken and added to AF2 cells. Stably transformed colonies were selected by puromycin resistance and confirmed by PCR analysis using the following primer set (Sense: 5'-TGCATGTTTGCCAGTTGATG-3 '(SEQ ID NO: 2); Antisense: 5 '-GCATGCCCATTGTTATCTGG-3' (SEQ ID NO: 3)).

Example 3: Toxicity Evaluation of Prodrugs in Breast Cancer Cells

To evaluate the effect of prodrugs on viability of breast cancer cells (MDA-MB-231; Korea Cell Bank, Korea) in vitro, MTT assay was performed. Incorporate 4.0 x 10 ³ cells / well of breast cancer cells in RPMI (PAA, Austria) medium 100 with 5 vol% bovine serum and dispense into 96-well plates and 37 ° C., 5% carbon dioxide and 90% Incubated for 24 hours under conditions of abnormal humidity. RPMI medium 100 with 5 vol% bovine serum was then added to each well and incubated after 24 hours and 72 hours 5-FC, 5-FU (Panel A of FIG. 3) or GCV (Sigma). -Aldrich, USA (Panel B of Figure 3) was treated for each concentration. On day 7, the MTT reagent (10 / ml) was treated and the absorbance was measured at 540 nm using an ELISA-reader. Absorbance values measured at this time were expressed as cell viability. Panel A of FIG. 3 shows cell viability when 5-FC and 5-FU were treated, and Panel B of FIG. 3 shows cell viability when GCV was treated, respectively. Apoptosis was not increased even at the maximum treatment concentrations of 5-FC (600 / mL) and GCV (4 / mL), and it was determined that 5-FC and GCV were not cytotoxic to breast cancer cells. On the other hand, in the experimental group treated with 5-FU, it was confirmed that as the concentration of 5-FU increased, the cell death rate increased. The significance of the experimental data was assessed using one-way ANOVA, and the significance level was calculated as 0.05 and marked as '*' when significance exists. These results prove that the administration of anticancer prodrugs 5-FC and GCV in vivo is safe because it does not show any toxicity.

Example  4: AF2 . CD - TK Effect of cancer cell death

Breast cancer cells were treated with AF2.CD-TK cells and prodrugs 5-FC and GCV together to confirm cell death effects. Incorporate 4.0 x 10 ³ cells / well of breast cancer cells in RPMI (PAA, Austria) medium 100 with 5 vol% bovine serum and dispense into 96-well plates and 37 ° C., 5% carbon dioxide and 90% Incubated for 24 hours under conditions of abnormal humidity. The pre-cultured AF2 cells and AF2.CD-TK cells were diluted to 8.0 × 10 ³ cells / well and distributed in 96-well plates in which breast cancer cells were cultured and incubated for 24 hours. Then after 24 and 72 hours 5-FU (500 / ml) and GCV (1 / ml) were treated. As a result, apoptosis was increased by 37% when AF2.CD-TK cells, 5-FC (500 / ml) and GCV (1 / ml) were treated together. As confirmed in the results shown in FIG. 3, 5-FC and GCV did not show cytotoxicity inducing apoptosis when treated with breast cancer cells, respectively, as shown in the results of FIG. 4. Cell death was increased when AF2.CD-TK cells were treated simultaneously. These results suggest that the CD and HSV-TK genes introduced into AF2.CD-TK cells converted prodrugs 5-FC and GCV into anticancer drugs, leading to cell death. In addition, compared to the experimental group treated with AF2.CD-TK cells and 5-FC or GCV separately, the synergistic effect was seen in the increased cell death in the experimental group treated with two prodrugs together. It confirmed that it represents. In addition, the experimental group treated with two prodrugs at the same time and the experimental group treated with 5-FC and treated with GCV after 24 hours, the cell death was further increased when the two prodrugs were treated simultaneously. In conclusion, it was demonstrated that AF2.CD-TK cells convert pro-drugs with no cytotoxicity to 5-FU and GCV-TP, which are potent cytotoxic substances, thereby exerting anticancer efficacy.

Example  5: Breast Cancer Animal Models AF2 . CD - TK Cancer treatment effect of cells

Breast Cancer Animal Model Production

Subcutaneous administration of 1.0 X 10 ⁶ breast cancer cell MDA-MB-231 to the mammary fat pad around the second papillae of the right mouse in 6-week-old immunodeficiency nude mice (BABL / c Nude). Induced breast cancer for 7 weeks to produce a breast cancer animal model. Tumor volume of breast cancer was measured by a vernier caliper and calculated by the formula (π / 6) × height × length × height (mm ³ ) for 7 weeks of tumor change, and the tumor volume was 250-300 mm ^3. Phosphorus animals were used for the experiment.

Labeled with fluorescent material AF2 . CD - TK  Cell fabrication

Chloromethyl Benzeamido-1, 1'-Dioctadecyl-3,3, 3 ', 3'-tetramethylindocarbocyanin perchlorate to investigate the location and role of AF2.CD-TK cells in animal models of breast cancer AF2.CD-TK cells were labeled with (Chloromethyl benzamido-1, 1′-dioctadecyl-3, 3, 3 ′, 3′-tetramethylindo carbocyanime perchlorate, CM-DiI, Sigma aldrich, USA). CM-DiI, a fluorescent material, was non-toxic and maintained for 10 weeks in the body, and was used as a cell marker. The method of labeling the cells with CM-DiI can be carried out using various protocols known in the art, preferably, the medium is removed by centrifugation, and the CM- remains only 4.0 × 10 ⁶ AF2.CD-TK cells. 1 ml (10 / ml) of DiI reagent was added and allowed to stand at 37 ° C, 5% carbon dioxide and humidity conditions, and after 15 minutes, centrifuged to remove the CM-DiI reagent and washed twice with phosphate buffer (PBS). It was. In fluorescence microscopy, CM-DiI labeled cells show red fluorescence.

Breast Cancer In Animal Models AF2 . CD - TK  Effect of cancer cell death by cells

The breast cancer animal model was divided into three groups: control group, 5-FU and AF2.CD-TK, 5-FU group was treated with 5-FU (50 mg / ml), and AF2.CD-TK group was AF2.CD-TK. Cells, 5-FC (500 mg / ml) and GCV (1 mg / ml) were treated. The AF2.CD-TK group was implanted with 4.0 × 10 ⁶ AF2.CD-TK cells labeled with CM-DiI, and three days later, 5-FC and GCV were intraperitoneally administered daily for 6 days. AF2.CD-TK cells labeled with CM-DiI were transplanted one more time after 15 days from the first cell transplantation, and three days thereafter, 5-FC and GCV were administered daily intraperitoneally for 6 days. The control group and the 5-FU group were administered with PBS (100) (control) or 5-FU (5-FU group) at the same time as the prodrugs of the AF2.CD-TK group (5-FC and GCV), respectively. . Thirty days after AF2.CD-TK cells were implanted in the AF2.CD-TK group, the animals of each experimental group were sacrificed and breast cancer tumors were collected and compared with vernier calipers. While this increased rapidly, tumor volume and weight in the AF2.CD-TK group were reduced by about 84% compared to the control (see C and D in FIG. 5). In the 5-FU group, tumor volume was reduced by 84% compared to the control group, but there were side effects such as diarrhea, systemic erythema, and death. In contrast, the AF2.CD-TK group effectively reduced tumor volume without the side effects caused by 5-FU.

Example  6: in breast cancer tissue AF2 . CD - TK  Cell identification

Tissue section  Slide authoring

AF2.CD-TK cells labeled with CM-DiI were subcutaneously administered around the breast cancer tissues of the mice, and 30 days later, the tumor tissues were collected from the mice, soaked in 10% formaldehyde and allowed to stand for at least 24 hours. Tumor tissue was fixed in a cassette (Hanil, Korea) and cut into appropriate sizes for dehydration. The dehydrated tissue was prepared using a paraffin holder (Leicar, Germany) using a paraffin block, and then sliced using a tissue slicer (Leicar, Germany) to make a slide.

Fluorescent staining

After removing paraffin from tumor tissue slides, 10% formaldehyde was fixed for 5 minutes and washed twice with PBS. Stained tissue cells were treated with 4 ', 6-diimino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI) for 10 minutes. DAPI is a fluorescent material that stains the nucleus of cells, which are displayed in blue on a fluorescence microscope. After staining, the plate was washed twice with PBS again and then properly drained, and then sealed using an encapsulant (Thermo, USA) and a cover glass. Slides were checked using a fluorescence microscope (Olympus, Japan) (Fig. 6). The blue part means tumor tissue stained with DAPI and the red part means AF2.CD-TK cells stained with CM-DiI. Panel A of FIG. 6 is a photograph of the outer layer of the tumor, and it can be seen that AF2.CD-TK cells gradually penetrate into the tumor from the outside of the tumor tissue. When the inside of the tumor was observed under a microscope, it was confirmed that AF2.CD-TK cells were present inside the tumor, and that the center of the tumor was empty. This results in AF2.CD-TK cells converting the prodrugs 5-FC and GCV to cytotoxic substances 5-FU and GCV-MP, where 5-FU and GCV-MP kill tumor cells and thereby An empty space appears to have occurred.

Hematoxylin-Eosin Staining

Hematoxylin-eosin standard staining method was used to compare breast skin tissues with mammary glands of each experimental group. Tissue section slides from which paraffin was removed were left standing in hematoxylin for 2 minutes, soaked in tap water for 15 minutes, and then left standing in Eosin for 3 minutes, dehydrated, and encapsulated using an encapsulant and a cover glass. As a result of the hematoxylin-eosin staining, the control showed a very invasion structure of breast cancer cells in the breast skin tissue, and the network structure of the dermal layer such as normal breast skin tissue (Normal) was found. None (see FIG. 7). On the other hand, AF2.CD-TK group showed a very similar structure to the normal breast skin tissue, and compared with the control group, the degree of breast cancer cell penetration into the breast skin tissue was hardly found (see FIG. 7). The breast skin tissues of the 5-FU group were similar to the control group, and normal breast skin tissues were destroyed because the 5-FU having strong cytotoxicity killed not only cancer cells but also healthy cells.

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. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Attach an electronic file to a sequence list

Claims (11)

delete delete delete delete An anticancer pharmaceutical composition for breast cancer comprising amniotic fluid-derived stem cells transformed with an expression vector comprising a nucleic acid molecule encoding a suicide gene as an active ingredient, wherein the suicide gene is cytosine deaminase. (cytosine deaminase, CD) and herpes simplex virus thymidine kinase (HSV-TK), and the amniotic fluid-derived stem cells are transformed with a vector expressing the v-myc oncogene to be immortalized. Stem cells, characterized in that the composition.
delete The method of claim 5, wherein the expression vector is a retroviral vector, baculovirus vector, adenovirus vector, Adeno-associated viruses (AAV) vector, lentivirus vector, herpes simplex virus vector or basin virus A composition, which is a vector.
6. The composition of claim 5, wherein the amniotic fluid-derived stem cells are stem cells derived from amniotic fluid of humans, pigs, horses, cattle, mice, rats, hamsters, rabbits, goats or sheep.
delete An anticancer kit for breast cancer comprising (i) amniotic fluid-derived stem cells transformed with an expression vector comprising a nucleic acid molecule encoding a suicide gene and expressing a suicide gene, and (ii) a prodrug of an anticancer agent. Are cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK), and the amniotic fluid-derived stem cells are transfected with vectors expressing the v-myc oncogene. Inverted immortalized stem cells, the anticancer prodrug is a kit, characterized in that 5-fluorocytosine (5-fluorocytosine) and ganciclovir.
delete

Images