PHARMACEUTICAL COMPOSITION FOR TREATING DRUG-RESISTANT CANCER COMPRISING EXOSOMES DERIVED FROM DIFFERENTIATING STEM CELLS AS AN ACTIVE INGREDIENT

Abstract

The present invention relates to a method of treating a subject having drug-resistant cancer, comprising administering a composition comprising exosomes derived from differentiating stem cells as an active ingredient. The exosomes isolated differentiating stem cells according to the present invention have an excellent expression rate of bioactive factors affecting differentiation and have an effect of facilitating reprogramming of cancer stem cells and differentiating them into cancer cells with weakened drug resistance.

Claims

1. A method of treating a subject having drug-resistant cancer, comprising administering a composition comprising exosomes isolated from differentiating stem cells as an active ingredient.

2. The method according to claim 1, wherein the differentiating stem cells are stem cells differentiating into osteocytes, adipocytes, cartilage cells, myocytes, fibroblasts, astrocytes or nerve cells.

3. The method according to claim 2, wherein the stem cells are marrow-derived stem cells, cord blood-derived stem cells or fat-derived stem cells.

4. The method according to claim 3, wherein the marrow-derived stem cells, cord blood-derived stem cells or fat-derived stem cells are human- or animal-derived stem cells.

5. The method according to claim 1, wherein the composition comprises exosomes at a concentration of 1 to 200 g/m.

6. The method according to claim 1, wherein the composition is administered simultaneously, separately or sequentially with radiation or an anticancer agent.

7. The method according to claim 6, wherein the anticancer agent is an antimetabolite, an alkylating agent, an anti-tumor antibiotic, a plant alkaloid, an antimitotic drug, a hormonal agent, or a platinum-containing compound.

8. The method according to claim 1, wherein the drug-resistant cancer is lung cancer, breast cancer, liver cancer, stomach cancer, colorectal cancer, colon cancer, skin cancer, bladder cancer, pancreatic cancer, prostate cancer, ovarian cancer, cervical cancer, thyroid cancer, renal cancer, fibrosarcoma, melanoma or hematologic cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG. 1 is a mimetic diagram of exosomes derived from human adipose-derived stem cells differentiating into osteocytes or adipocytes according to one example of the present invention and its applications.

[0081] FIG. 2 is a drawing which shows the result of analysis of characteristics of exosomes (D-Exo) isolated from human adipose-derived stem cells differentiating into osteocytes according to one example of the present invention, and is a drawing which shows (A) the structure and shape of the exosomes determined by using a transmission electron microscope, and (B) the size of exosomes determined by using a dynamic light scattering. Herein, exosomes (P-Exo) isolated from human adipose-derived stem cells are used as a control group and the scale bar represents 50 nm.

[0082] FIG. 3 is a drawing which shows the result of inducing differentiation of human adipose-derived stem cells into osteocytes by treating exosomes (D-Exo) isolated from stem cells which is differentiating into osteocytes for 21 days by concentration (5 g/m, 10 g/m, 20 g/m, 50 g/m, and 100 g/m) according to one example of the present invention, and is a drawing which shows (A) the result of Alizarin red s staining, and (B) the calcium detection assay result measuring the absorbance at 562 nm by de-staining the Alizarin red s stained stem cells using 10% cetylpyridinium chloride. Herein, DM is differentiation medium, and P-Exo is the exosome isolated from proliferating human adipose-derived stem cells, and GM is cell culture medium (growth medium).

[0083] FIG. 4 is a drawing which shows the result of inducing differentiation of bone cancer stem cells into osteocytes by treating exosomes (D-Exo) isolated from stem cells which is differentiating into osteocytes to CD133+ bone cancer stem cells (CD133+MG63) for 14 days by concentration (5 g/m, 10 g/m, and 30 g/m) according to one example of the present invention, and is (A) a microscopic observation photograph of bone cancer stem cells after treating exosomes (D-Exo) isolated from stem cells differentiating into osteocytes for 14 days by concentration (5 g/m, 10 g/m, and 30 g/m, and (B) a graph showing the relative change of osteocyte differentiation genes, ALPL (Alkaline phosphatase), BGLAP (Osteocalcin), RUNX2 (Runt-related transcription factor 2) and COL1A1 (Collagen, type I, alpha 1), after treating exosomes (D-Exo) isolated from stem cells differentiating into osteocytes for 14 days by concentration (5 g/m, 10 g/m, and 30 g/m). Herein, GM is cell culture medium (growth medium), and D-Exo is the exosome isolated from differentiating stem cells from human adipose-derived stem cells into osteocytes.

[0084] FIG. 5 is a drawing which shows the expression rate of osteocyte differentiation genes by performing Human Osteogenesis PCR array analysis according to one example of the present invention, and is (A) a graph showing the relative gene expression rate of the group in which the cell culture medium (GM, negative control) is treated and the group in which exosomes (D-Exo) isolated from stem cells which is differentiating into osteocytes are treated, to bone cancer stem cells for 14 days, and (B) a table showing osteocyte differentiation-related genes overexpressed in the group in which exosomes (D-Exo) isolated from stem cells differentiating into osteocytes are treated, compared to the negative control group in which the cell culture medium (GM, growth medium) is treated. Herein, GM is cell culture medium (growth medium), and D-Exo is the exosome isolated from differentiating stem cells from human adipose-derived stem cells into osteocytes.

[0085] FIG. 6 is a drawing which shows the expression rate of drug resistance genes using PCR array according to one example of the present invention, and is a drawing which shows (A) the array result showing the relative gene expression rate of the group in which exosomes (D-Exo) isolated from stem cells differentiating into osteocytes for 14 days are treated to bone cancer stem cells, and the group in which the exosomes (D-Exo) are not treated, and (B) a table digitizing the relative gene expression rate.

[0086] FIG. 7 is a drawing showing the relative gene expression rate of a drug transporter according to one example of the present invention. Herein, CSC is the group in which exosomes (D-Exo) isolated from differentiating stem cells from bone cancer stem cells into osteocytes are not treated, and CSC+D-Exo is the group in which exosomes (D-Exo) isolated from differentiating stem cells from bone cancer stem cells into osteocytes are treated to bone cancer stem cells for 14 days.

DETAILED DESCRIPTION OF THE INVENTION

[0087] Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention only, but the scope of the present invention is not limited by these examples.

EXAMPLES

Example 1: Isolation of Exosomes from Stem Cells Differentiating into Osteocytes or Adipocytes

[0088] To isolate each exosome from stem cells differentiating into osteocytes or adipocytes, human adipose-derived stem cells sub-cultured by passages 3 to 7 were cultured in osteocyte differentiation medium (DMEM high concentration glucose (Dulbecco Modified Eagle Medium) comprising 10% fetal bovine serum, 1% penicillin/streptomycin, 1 M dexamethasone, 0.5 mM ascorbic acid and 0.01 M -glycerophosphate or adipocyte differentiation medium (DMEM high concentration glucose (Dulbecco's Modified Eagle's Medium high glucose) comprising 5% fetal bovine serum, 1 M dexamethasone, 1 g/m insulin, 100 M indomethacin and 0.5 mM 3-isobutyl-1-methylxanthine, respectively, and were maintained for 24 hours by replacing it with DMEM medium that was serum-free and antibiotic-free medium without phenol red for 24 hours once per 3 days. After 24 hours, the culture supernatant of differentiating stem cells was collected to isolate exosomes (D-Exo).

[0089] The collected cell culture supernatant was centrifuged at 300g for 5 minutes to remove cells, and was centrifuged at 10,000g for 30 minutes to remove cell secretions. Then, it was centrifuged at 100,000g for 70 minutes using an ultracentrifuge to obtain exosome precipitates. After this, it was washed at 100,000g for 70 minutes twice in total by adding phosphate-buffered saline (PBS). The washed exosome precipitates were resuspended in phosphate-buffered saline.

Comparative Example 1: Isolation of Exosomes (P-Exo) from Proliferating Stem Cells

[0090] In order to compare with the efficacy of exosomes derived from stem cells differentiating into osteocytes or adipocytes, exosomes (P-Exo) were isolated from proliferating human adipose-derived stem cells as a comparative control group from the supernatant obtained by culturing human adipose-derived stem cells sub-cultured by passages 3 to 7 in common culture medium (DMEM (Dulbecco Modified Eagle Medium) comprising 10% fetal bovine serum and 1% penicillin/streptomycin, and then maintaining them for 24 hours by replacing it with DMEM medium that was serum-free and antibiotic-free medium without phenol red.

[0091] The collected cell culture supernatant was centrifuged at 300g for 5 minutes to remove cells, and was centrifuged at 10,000g for 30 minutes to remove cell secretions. Then, it was centrifuged at 100,000g for 70 minutes using an ultracentrifuge to obtain exosome precipitates. After this, it was washed at 100,000g for 70 minutes twice in total by adding phosphate-buffered saline (PBS). The washed exosome precipitates were resuspended in phosphate-buffered saline.

Example 2: Analysis of Characteristics of Exosomes (D-Exo) Isolated from Stem Cells Differentiating into Osteocytes

[0092] The size and shape of the exosomes derived from Example 1 and Comparative example 1 were confirmed using a transmission electron microscope and a dynamic light scattering.

[0093] As a result, the shape of each exosome isolated could be confirmed by the transmission electron microscope (A of FIG. 2), and the size of exosomes was confirmed as the size of exosomes (D-Exo) isolated from stem cells differentiating into osteocytes was about 78.52 nm on average, and the size of exosomes (P-Exo) isolated from proliferating stem cells was about 87.17 nm on average (B of FIG. 2).

Example 3: Osteocyte Differentiation Induction of Human Adipose-Derived Stem Cells Using Exosomes (D-Exo) Isolated from Stem Cells Differentiating into Osteocytes

[0094] To confirm the differentiation-inducing ability of exosomes (D-Exo) isolated from stem cells differentiating into osteocytes from human adipose-derived stem cells, a medium composition comprising exosomes derived from stem cells differentiating into osteocytes was used. Here, a medium composition comprising exosomes (P-Exo) isolated from proliferating stem cells was used as a comparative control group. The medium composition was used by adding exosomes (D-Exo) isolated from stem cells differentiating into osteocytes at a concentration of 5 g/m, 10 g/m, 20 g/m, 50 g/m, and 100 g/m to common culture medium (DMEM (Dulbecco Modified Eagle Medium) comprising 10% fetal bovine serum and 1% penicillin/streptomycin). After treating the medium compositions to human adipose-derived stem cells (hASCs), respectively, the medium compositions were replaced once per 3 days for 14 days. Then, as a negative control group (Growth medium, GM), stem cells cultured in DMEM high concentration glucose (Dulbecco's Modified Eagle's Medium high glucose) medium comprising 10% fetal bovine serum and 1% penicillin/streptomycin were used, and as a positive control group (Differentiation medium, DM), stem cells cultured in DMEM high concentration glucose (Dulbecco's Modified Eagle's Medium high glucose) medium comprising 10% fetal bovine serum, 1% penicillin/streptomycin, 1 M dexamethasone, 0.5 mM ascorbic acid, and 0.01 M -glycerophosphate were used.

[0095] Then, for stem cells in which differentiation into osteocytes was induced for 14 days, Alizarin red s staining was used to analyze whether cells were differentiated. In addition, Alizarin red s stained stem cells were de-stained using 10% cetylpyridinium chloride and the absorbance at 562 nm was measured, thereby quantifying the degree of differentiation.

[0096] As a result, it could be confirmed that when treating exosomes (D-Exo) isolated from stem cells differentiating into osteocytes at a concentration of 10 g/m or more for 14 days, osteocyte differentiation was induced at a similar level to the positive control group. On the other hand, it was confirmed that in the case of stem cells treated with exosomes (P-Exo) isolated from proliferating stem cells, they did not differentiate into osteocytes, but only proliferation was achieved (FIG. 3).

Example 4: Bone Cancer Cell Differentiation Induction of Bone Cancer Stem Cells Using Exosomes (D-Exo) Isolated from Stem Cells Differentiating into Osteocytes and Analysis of Expression Rate of Osteocyte Differentiation Genes Using PCR Array

[0097] To confirm the differentiation-inducing ability of exosomes (D-Exo) isolated from stem cells differentiating into osteocytes from human adipose-derived stem cells, a medium composition comprising exosomes derived from stem cells differentiating into osteocytes were used. The medium composition was used by adding exosomes (D-Exo) isolated from stem cells differentiating into osteocytes at a concentration of 5 g/m, 10 g/m, and 30 g/m to common culture medium (DMEM (Dulbecco Modified Eagle Medium) comprising 10% fetal bovine serum and 1% penicillin/streptomycin). The medium composition was treated to cultured CD133+ bone cancer stem cells (CD133+MG63), respectively, and then the medium composition was replaced once per 3 days for 14 days. Then, as a negative control group (Growth medium, GM), stem cells cultured in DMEM high concentration glucose (Dulbecco's Modified Eagle's Medium high glucose) comprising 3% fetal bovine serum and 1% penicillin/streptomycin were used.

[0098] After this, for bone cancer stem cells in which differentiation into osteocytes was induced for 14 days, whether cells were differentiated was analyzed using a PCR analysis technique.

[0099] As a result, it was confirmed that when treating exosomes (D-Exo) isolated from stem cells differentiating into osteocytes at a concentration of 30 g/m or more for 14 days, osteocyte differentiation genes such as ALPL (Alkaline phosphatase), BGLAP (Osteocalcin), RUNX2 (Runt-related transcription factor 2), etc. were expressed 2 to 45 times higher than the negative control group (FIG. 4).

[0100] In addition, as the result of qualifying the mRNA expression rate through Human Osteogenesis PCR array analysis, it was confirmed that 27 genes of 73 kinds of osteocyte differentiation-related genes in total were expressed over 2 times higher in the group in which exosomes (D-Exo) isolated from stem cells differentiating into osteocytes were treated (FIG. 5).

Example 5: Analysis of Expression Rate of Drug Resistance Gene Using PCR Array and Analysis of Relative Gene Expression Rate of Drug Transporter

[0101] To confirm the bone cancer stem cell drug resistance inhibitory ability of exosomes (D-Exo) isolated from stem cells differentiating into osteocytes from human adipose-derived stem cells, a medium composition comprising exosomes (D-Exo) isolated from stem cells which were differentiating into osteocytes was used. The medium composition was used by adding exosomes derived from stem cells differentiating into osteocytes at a concentration of 30 g/m to common culture medium (DMEM (Dulbecco Modified Eagle Medium) comprising 10% fetal bovine serum and 1% penicillin/streptomycin). The medium composition was treated to the cultured CD133+ bone cancer stem cells (CD133+MG63), and then the medium composition was replaced once per 3 days for 14 days. Then, as a negative control group (Growth medium, GM), stem cells cultured in DMEM high concentration glucose (Dulbecco's Modified Eagle's Medium high glucose) medium comprising 3% fetal bovine serum and 1% penicillin/streptomycin were used.

[0102] After this, for bone cancer stem cells in which differentiation into osteocytes was induced for 14 days, whether cells were differentiated was analyzed using a PCR analysis technique.

[0103] As a result, it was confirmed that 64 genes among 84 kinds of drug resistance-related genes in total were inhibited in the group in which exosomes (D-Exo) isolated from stem cells differentiating into osteocytes were treated (A of FIG. 6). In particular, it was confirmed that APC, BLMH, BRCA1, BRCA2, DHFR, MSH2, NAT2, RARB, and XPC genes were inhibited 10 to 415 times (B of FIG. 6).

[0104] In addition, the expression of ABCC1, ABCC5, ABCG2, and MVP genes among drug transporter genes which directly affected drug resistance was significantly lowered (FIG. 7).