USE OF ADENOSINE DIPHOSPHATE RIBOSE FOR ADJUVANT THERAPY WITH RADIATION AND/OR ANTI-CANCER TREATMENT

Abstract

The present disclosure relates to an anti-cancer therapy using adenosine diphosphate ribose (ADP-Ribose), and specifically, to a single anti-cancer therapy of ADP-ribose and a combination therapy of ADP-ribose with an anti-cancer drug or radiation, and the like.

Claims

1. An anti-cancer composition comprising: adenosine diphosphate ribose (ADP-ribose) or a pharmaceutically acceptable salt thereof.

2. The anti-cancer composition of claim 1, wherein the cancer is at least one solid cancer selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, kidney cancer, stomach cancer, and ovarian cancer.

3. The anti-cancer composition of claim 1, wherein the cancer is a metastatic cancer.

4. The anti-cancer composition of claim 1, wherein the composition causes disturbance of biochemical action in cancer cells by inducing accumulation of ADP-ribose in cancer cells.

5. An anti-cancer adjuvant for radiation therapy comprising: adenosine diphosphate ribose (ADP-ribose) or a pharmaceutically acceptable salt thereof.

6. The anti-cancer adjuvant of claim 5, which improves radiation sensitivity.

7. An anti-cancer composition comprising: (i) adenosine diphosphate ribose (ADP-ribose) or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer drug.

8. The anti-cancer composition of claim 7, wherein the second anti-cancer drug is at least one selected from the group consisting of a cytotoxic anti-cancer drug, a targeted anti-cancer drug, and an immuno-oncology drug.

9. The anti-cancer composition of claim 8, wherein the second anti-cancer drug is the cytotoxic anti-cancer drug.

10. The anti-cancer composition of claim 9, wherein the cytotoxic anti-cancer agent is an alkylating agent, and the ADP-ribose or a pharmaceutically acceptable salt thereof enhances sensitivity to the alkylating agent.

11. The anti-cancer composition of claim 8, wherein the second anti-cancer drug is the targeted anti-cancer drug.

12. The anti-cancer composition of claim 11, wherein the targeted anti-cancer drug targets at least one target selected from the group consisting of VEGF/VEGFR, EGFR, and HER2, and the ADP-ribose or a pharmaceutically acceptable salt thereof enhances sensitivity to the targeted anti-cancer drug.

13. The anti-cancer composition of claim 12, wherein the targeted anti-cancer drug is a VEGF/VEGFR inhibitor for the treatment of brain or liver cancer.

14. The anti-cancer composition of claim 12, wherein the targeted anti-cancer drug is an EGFR inhibitor for the treatment of lung cancer.

15. The anti-cancer composition of claim 12, wherein the targeted anti-cancer drug is a HER2 inhibitor for the treatment of breast cancer.

16. The anti-cancer composition of claim 8, wherein the second anti-cancer drug is the immuno-oncology drug.

17. An anti-cancer adjuvant for chemotherapy comprising: adenosine diphosphate ribose (ADP-ribose) or a pharmaceutically acceptable salt thereof.

18. The anti-cancer adjuvant of claim 17, wherein an anti-cancer drug therapy and the anti-cancer adjuvant are administered at the same time or at different times.

19. A method for preventing or treating cancer, the method comprising administering anti-cancer composition according to claim 1 to a subject in need thereof.

20. A method for preventing or treating cancer, the method comprising administering anti-cancer composition according to claim 7 to a subject in need thereof.

Description

DESCRIPTION OF DRAWINGS

[0092] FIG. 1 shows the result of confirming the accumulation of ADP-ribose in various solid cancer cells after ADP-ribose treatment on the cells (A: U-87MG, B: H1975, C: AsPC-1, D: Hep G2, E: MDA-MB-231, F: HCT116, and G: Caki-1 cell line).

[0093] FIG. 2 is a photomicrograph (A) and a graph (B) each confirming the cancer cell growth inhibition and cell death effect after ADP-ribose treatment on brain cancer (U-87MG) cells.

[0094] FIG. 3 is a photomicrograph (A) and a graph (B) confirming the cancer cell growth inhibition and cell death effect after ADP-ribose treatment on lung cancer (H1975) cells.

[0095] FIG. 4 is a photomicrograph (A) and a graph (B) confirming the cancer cell growth inhibition and cell death effect after ADP-ribose treatment on pancreatic cancer (AsPC-1) cells.

[0096] FIG. 5 is a photomicrograph (A) and a graph (B) confirming the cancer cell growth inhibition and cell death effect after ADP-ribose treatment on liver cancer (Hep G2) cells.

[0097] FIG. 6 is a photomicrograph (A) and a graph (B) confirming the cancer cell growth inhibition and cell death effect after ADP-ribose treatment on breast cancer (MDA-MB-231) cells.

[0098] FIG. 7 is a photomicrograph (A) and a graph (B) confirming the cancer cell growth inhibition and cell death effect after ADP-ribose treatment on colon cancer (HCT116) cells.

[0099] FIG. 8 is a photomicrograph (A) and a graph (B) confirming the cancer cell growth inhibition and cell death effect after ADP-ribose treatment on kidney cancer (Caki-1) cells.

[0100] FIG. 9 is a graph confirming the cancer cell death effect after ADP-ribose treatment on gastric cancer (SNU-1) cells or ovarian cancer (OVCAR-3) cells.

[0101] FIG. 10 is a photomicrograph (A) and a graph (B) confirming the reduction in cancer cell invasion and metastasis ability after ADP-ribose treatment of pancreatic cancer (AsPC-1) cells.

[0102] FIG. 11 is a photomicrograph (A) and a graph (B) confirming the cancer cell invasion and metastasis ability after ADP-ribose treatment on breast cancer (MDA-MB-231) cells.

[0103] FIG. 12 is a graph (A) and a photomicrograph (B) showing the effect of reducing tumor volume after subcutaneous injection (S.C) of ADP-ribose in a xenograft animal model induced with a pancreatic cancer cell line.

[0104] FIG. 13 is a graph (A) and a photomicrograph (B) showing the effect of reducing tumor volume after intravenous injection (I.V) of ADP-ribose in a xenograft animal model induced with a kidney cancer cell line.

[0105] FIG. 14 is a graph (A) and a photomicrograph (B) showing the effect of reducing tumor volume after oral administration (P.O) of ADP-ribose in a xenograft animal model induced with a pancreatic cancer cell line.

[0106] FIG. 15 is a graph showing the synergistic cell death effect when various solid tumor cells (A: U-87MG, B: H1975, C: AsPC-1, D: Hep G2, E: MDA-MB-231, and F: HCT116) were treated in combination with low-dose anti-cancer drugs (A: bevacizumab, B: osimertinib, C: gemcitabine, D: sorafenib, E: Herceptin, and F: 5-fluorouracil) and ADP-ribose.

[0107] FIG. 16 is a graph showing the synergistic tumor volume reduction effect when tumor-inducing animals were administered in combination with low-dose anti-cancer drugs (A: bevacizumab, B: osimertinib, C: gemcitabine, and D: sorafenib) and ADP-ribose.

[0108] FIG. 17 is a graph showing the comparison results of ADP-ribose accumulation in cells when solid cancer (A: U-87MG, B: Caki-1, C: AsPC-1, and D: MDA-MB-231) cells were irradiated with radiation and when treated with ADP-ribose.

[0109] FIG. 18 is a graph confirming the synergistic cell death effect when solid cancer (A: U-87MG, B: H1975, C: AsPC-1, D: Hep G2, E: MDA-MB-231, F: HCT116, and G: Caki-1) cells were treated in combination with the irradiation-resistant dose of radiation and ADP-ribose.

[0110] FIG. 19 is a graph showing the synergistic tumor volume reduction effect when tumor-inducing animals were administered in combination with low-dose irradiation and ADP-ribose.

[0111] FIG. 20 is a photograph and a graph confirming whether cytotoxicity was observed after ADP-ribose was administered to normal cells (A, B: human colon fibroblast, and C, D: human hair dermal papilla cells).

MODE FOR INVENTION

[0112] Hereinafter, the present disclosure will be described in more detail through Examples. However, these Examples are provided to illustrate the present disclosure by way of example, and the scope of the present disclosure is not limited to these Examples.

[0113] Materials and Methods

[0114] 1. Preparation of ADP-Ribose and Anti-Cancer Drug

[0115] ADP-ribose and anti-cancer drugs such as bevacizumab, osimertinib, gemcitabine, sorafenib, herceptin, and 5-fluorouracil were all purchased from Sigma-Aldrich (St. Louis, MO, USA). ADP-ribose and all anti-cancer drugs were stored frozen at −20° C. until use.

[0116] 2. Preparation of Cell Lines

[0117] Brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (Aspc-1), liver cancer (Hep G2), breast cancer (MDA-MB-231), colon cancer (HCT116), kidney cancer (Caki-1), gastric cancer (SNU-1) and ovarian cancer (OVCAR-3) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and stored in liquid nitrogen until used for research.

[0118] 3. ADP-Ribose Quantitative Analysis

[0119] 5×10.sup.5 brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (Aspc-1), liver cancer (Hep G2), breast cancer (MDA-MB-231), colon cancer (HCT116), and kidney cancer (Caki-1) cells were mixed with 10% fetal calf serum+100 units/ml penicillin+100 μg/ml streptomycin in each appropriate medium, and the cells were divided into the untreated group and ADP-ribose-treated groups, and cultured under conditions of 37° C. and 5% CO.sub.2 for 24 hours. After culturing, the culture medium was removed, the cells were washed with phosphate-buffered saline, and then RIPA buffer was added and the cells were left on ice for 20 minutes. After scraping the cells, the cells were put in a centrifuge tube and centrifuged for 10 minutes at 10,000 g and 4° C. The supernatant was obtained, and 1% SDS was added, and then the reaction product was boiled at 100° C. for 5 minutes and cooled on ice. The supernatant was centrifuged again at 10,000 g and 4° C. for 10 minutes to obtain the final supernatant, and ADP-ribose was quantitatively analyzed and compared using an ELISA analysis kit (Cell Biolabs, Inc., San Diego, CA, USA).

[0120] 4. Cancer Cell Death Verification

[0121] 3×10.sup.3 Brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (Aspc-1), liver cancer (Hep G2), breast cancer (MDA-MB-231), colon cancer (HCT116), kidney cancer (Caki-1), gastric cancer (SNU-1) and ovarian cancer (OVCAR-3) cells were cultured in a 96-well plate under conditions of 37° C. and 5% CO.sub.2 for 24 hours, and then each cancer cell was treated with ADP-ribose at each concentration. After culturing for 24 hours under conditions of 37° C. and 5% CO.sub.2 again, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to determine the concentration of half-lethality and maximum lethality compared to untreated cells.

[0122] 5. Verification of Inhibition of Metastasis and Invasion Ability of Cancer Cell

[0123] Metastasis and invasion ability of cancer cells were analyzed by using a transwell having polycarbonate membrane filters with 8.0 μm pore size. The transwell was put into a 24-well plate. In each well, pancreatic cancer (Aspc-1) and breast cancer (MDA-MB-231) cells were seeded on the upper surface of the insert at a density of 3.5×10.sup.5 cells per well, divided into the untreated group and ADP-ribose-treated groups, and cultured under conditions of 37° C. and 5% CO.sub.2 for 24 hours. After culturing, the transwell was carefully washed with sterile water and then placed in 100% methanol to fix the attached cells. After fixation, the cells were stained with hematoxylin and an eosin reagent to observe the cells, and the ratio of cells that permeated the upper surface of the insert and metastasized and invaded the lower surface, compared to the untreated group, was obtained.

[0124] 6. Preparation of Animals

[0125] 5-week-old BALB/c nude mice were ordered from Charles River Laboratories (Wilmington, MA, USA). The breeding facility was an environment without specific lesions and maintained at a temperature of 22-25° C. the day and night cycle was maintained in a 12-hour cycle (lights on at 8:00 am), and feed and drinking water were provided autonomously.

[0126] All animal studies were conducted according to the regulations of the Animal Research Ethics Committee (AREC).

[0127] 7. Verification of Anti-Cancer Efficacy According to the Administration Route and Concentration of ADP-Ribose in the Xenograft Model

[0128] Pancreatic cancer (AsPC-1) or kidney cancer (Caki-1) cells (1×10.sup.7 cells) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were grown up to approximately 150 mm.sup.3 of the tumor volume. Then, three concentrations of ADP-ribose were administered subcutaneously, intravenously, and orally 3 times a week. The tumor size was measured every 6 days using a digital caliper, and the changes in tumor volume were compared for each group.

[0129] 8. Verification of Cell Death by Combination Treatment with Anti-Cancer Drug in Cancer Cell

[0130] 3×10.sup.3 Brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (Aspc-1), liver cancer (Hep G2), breast cancer (MDA-MB-231), and colon cancer (HCT116) cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% Ca.sub.2, respectively. Then, the brain cancer (U-87MG) cells were treated with 15 mM bevacizumab, the lung cancer (H1975) cells were treated with 1 nM osimertinib, pancreatic cancer (Aspc-1) cells were treated with 1 μM gemcitabine, the liver cancer (Hep G2) cells were treated with 1 μM sorafenib, the breast cancer (MDA-MB-231) cells were treated with 2.5 μM herceptin, and the colon cancer (HCT116) cells were treated with 10 μM 5-fluorouracil alone or in combination with ADP-ribose. After the treatment was terminated, the cells were cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to compare the degree of cell death.

[0131] 9. Verification of Anti-Cancer Efficacy According to the Combination of Anti-Cancer Drugs in the Xenograft Model

[0132] Brain (U-87MG), lung (H1975), pancreatic (AsPC-1), or liver (Hep G2) cells (1×10.sup.7 cells) were inoculated into 5-week-old BALB/c nude mice on the back flank, respectively. Then, when tumors were grown to a volume of approximately 150 mm.sup.3, the mice were divided into the control group (control), an anti-cancer drug alone treatment group, and a combination treatment group. To mice, for the brain cancer (U-87MG) study, 25 mg/kg low-dose of bevacizumab was administered by intraperitoneal injection twice a week; for lung cancer (H1975) study, 1 mg/kg low-dose of osimertinib was administered by oral injection twice a week; for pancreatic cancer (AsPC-1) study, 50 mg/kg low-dose gemcitabine was administered by intraperitoneal injection twice a week; and for liver cancer (Hep G2) study, 10 mg/kg low dose of sorafenib was administered by oral injection 5 times a week either alone or in combination with each of the above-described anti-cancer drugs and 10 mg/kg of ADP-ribose (subcutaneous administration, 3 times a week). The tumor size was measured every 6 days using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0133] 10. Quantitative Analysis of ADP-Ribose According to the Combination Treatment of Radiation in Cancer Cells

[0134] Brain cancer (U-87MG), kidney cancer (Caki-1), pancreatic cancer (AsPC-1), or breast cancer (MDA-MB-231) cells were cultured in a 6-well plate, respectively, and the cells were divided into the untreated group, the radiation-treated group, and the ADP-ribose-treated group. Irradiation was performed using an X-Rad 320 irradiator (Precision X-ray, North Branford, CT, USA), and cancer cells were treated with a total of 1, 2, and 5 Gy at a dose rate of 300 kVp and 150 cGy/min using an X-ray beam filter composed of 12.5 mA and 2.0 mm Al, respectively. Before treatment and at 4, 8, 16, and 24 hours after treatment, the medium was removed, the cells were washed with phosphate-buffered saline, and then RIPA buffer was added and the products were left on ice for 20 minutes. After scraping the cells, the cells were put in a centrifuge tube and centrifuged for 10 minutes at 10,000 g and 4° C. The supernatant was obtained, and 1% SDS was added, and then the reaction product was boiled at 100° C. for 5 minutes and cooled on ice. The supernatant was centrifuged again at 10,000 g and 4° C. for 10 minutes to obtain the final supernatant, and ADP-ribose was quantitatively analyzed using an ELISA analysis kit (Cell Biolabs, Inc., San Diego, CA, USA).

[0135] 11. Verification of Cell Death According to the Combination Treatment of Radiation in Cancer Cells

[0136] 3×10.sup.3 Brain cancer (U-87MG), lung cancer (H1975), pancreatic cancer (Aspc-1), liver cancer (Hep G2), breast cancer (MDA-MB-231), colon cancer (HCT116), and kidney cancer (Caki-1) cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose or 2 or 5 Gy radiation alone or in combination with ADP-ribose and 2 Gy radiation, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to compare the degree of cell death.

[0137] 12. Verification of Anti-Cancer Efficacy According to the Combination Treatment of Radiation in the Xenograft Model

[0138] Brain cancer (U-87MG), pancreatic cancer (AsPC-1), breast cancer (MDA-MB-231), or kidney cancer (Caki-1) cells (1×10.sup.7 cells) were inoculated into 5-week-old BALB/c nude mice on the back flank, respectively. Then, when tumors were grown to a volume of approximately 150 mm.sup.3, the mice were divided into three groups: the control group (Control), 2 Gy dose of radiation alone, and a combination administration of 2 Gy dose of radiation+10 mg/kg of ADP-ribose (subcutaneous administration, 3 times a week). The irradiation was performed with an X-Rad 320 irradiator (Precision X-ray, North Branford, CT, USA), wherein the mice were placed by maintaining an appropriate distance from the radiation source for irradiation with a predetermined radiation dose. A lead shield was used to protect the body parts or organs from exposure to radiation, other than the cancerous part of the mouse. The irradiation was performed with a total of 2 Gy of radiation fractionated into two doses of 1 Gy each (for 2 days) at a dose rate of 300 kVp and 150 cGy/min using an X-ray beam filter composed of 12.5 mA and 2.0 mm Al. The tumor size was measured every 6 days using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0139] 13. Statistical Significance Analysis

[0140] All graphs are presented as mean±standard deviation values. Statistical significance was analyzed using Student's t-test analysis according to whether data were normalized or not. A difference of P<0.05 or more was considered statistically significant, and Systat Software's program was used (Sigmastat ver. 3.5, Systat Software Inc., Chicago, IL, USA) for statistical significance analysis.

Example 1: Change in ADP-Ribose Level in Cancer Cells

Example 1-1: Change in ADP-Ribose Level in U-87MG Cells

[0141] 5×10.sup.5 U-87MG cells were divided into the untreated group and 8 μM ADP-ribose-treated group and cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% Fetal calf serum+100 units/ml penicillin+100 μg/ml streptomycin for 24 hours under conditions of 37° C. and 5% CO.sub.2. The medium of the cultured cells was removed, and the supernatant prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS) was used for ELISA analysis to detect and analyze ADP-ribose.

[0142] As a result, it was confirmed that when brain cancer cells (U-87MG) were treated with ADP-ribose, the amount of ADP-ribose remarkably increased significantly (P<0.001) compared to the control group (FIG. 1A).

Example 1-2: Change in ADP-Ribose Level in H1975 Cells

[0143] 5×10.sup.5 H1975 cells were divided into the untreated group and 4 μM ADP-ribose-treated group and cultured in RPMI-1640 medium supplemented with 10% fetal calf serum+100 units/ml penicillin+100 μg/ml streptomycin for 24 hours under conditions of 37° C. and 5% CO.sub.2. The medium of the cultured cells was removed, and the supernatant prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS) was used for ELISA analysis to detect and analyze ADP-ribose.

[0144] As a result, it was confirmed that when lung cancer cells (H1975) were treated with ADP-ribose, the amount of ADP-ribose remarkably increased significantly (P<0.001) compared to the control group (FIG. 1B).

Example 1-3: Change in ADP-Ribose Level in AsPC-1 Cells

[0145] 5×10.sup.5 AsPC-1 cells were divided into the untreated group and 8 μM ADP-ribose-treated group and cultured in RPMI-1640 medium supplemented with 10% fetal calf serum+100 units/ml penicillin+100 μg/ml streptomycin for 24 hours under conditions of 37° C. and 5% CO.sub.2. The medium of the cultured cells was removed, and the supernatant prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS) was used for ELISA analysis to detect and analyze ADP-ribose.

[0146] As a result, it was confirmed that when pancreatic cancer cells (AsPC-1) were treated with ADP-ribose, the amount of ADP-ribose remarkably increased significantly (P<0.001) compared to the control group (FIG. 1C).

Example 1-4: Change in ADP-Ribose Level in Hep G2 Cells

[0147] 5×10.sup.5 Hep G2 cells were divided into the untreated group and 8 μM ADP-ribose-treated group and cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum+100 units/ml penicillin+100 μg/ml streptomycin under conditions of 37° C. and 5% CO.sub.2 for 24 hours. The medium of the cultured cells was removed, and the supernatant prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS) was used for ELISA analysis to detect and analyze ADP-ribose.

[0148] As a result, it was confirmed that when liver cancer cells (Hep G2) were treated with ADP-ribose, the amount of ADP-ribose remarkably increased significantly (P<0.001) compared to the control group (FIG. 1D).

Example 1-5: Change in ADP-Ribose Level in MDA-MB-231 Cells

[0149] 5×10.sup.5 MDA-MB-231 cells were divided into the untreated group and 16 μM ADP-ribose-treated group and cultured in Leibovitz's L-15 medium supplemented with 10% fetal calf serum+100 units/ml penicillin+100 μg/ml streptomycin under conditions of 37° C. and 5% CO.sub.2 for 24 hours. The medium of the cultured cells was removed, and the supernatant prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS) was used for ELISA analysis to detect and analyze ADP-ribose.

[0150] As a result, it was confirmed that when breast cancer cells (MDA-MB-231) were treated with ADP-ribose, the amount of ADP-ribose remarkably increased significantly (P<0.001) compared to the control group (FIG. 1E).

Example 1-6: Change in ADP-Ribose Level in HCT116 Cell

[0151] 5×10.sup.5 HCT116 cells were divided into the untreated group and 8 μM ADP-ribose-treated group and cultured in McCoy's 5A medium supplemented with 10% fetal calf serum+100 units/ml penicillin+100 μg/ml streptomycin for 24 hours under conditions of 37° C. and 5% CO.sub.2. The medium of the cultured cells was removed, and the supernatant prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS) was used for ELISA analysis to detect and analyze ADP-ribose.

[0152] As a result, it was confirmed that when colon cancer cells (HCT116) were treated with ADP-ribose, the amount of ADP-ribose remarkably increased significantly (P<0.001) compared to the control group (FIG. 1F).

Example 1-7: Change in ADP-Ribose Level in Caki-1 Cell

[0153] 5×10.sup.5 Caki-1 cells were divided into the untreated group and 16 μM ADP-ribose-treated group and cultured in McCoy's 5A medium supplemented with 10% fetal calf serum+100 units/ml penicillin+100 μg/ml streptomycin for 24 hours under conditions of 37° C. and 5% CO.sub.2. The medium of the cultured cells was removed, and the supernatant prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS) was used for ELISA analysis to detect and analyze ADP-ribose.

[0154] As a result, it was confirmed that when kidney cancer cells (Caki-1) were treated with ADP-ribose, the amount of ADP-ribose remarkably increased significantly (P<0.001) compared to the control group (FIG. 1G).

[0155] Summarizing the above, Example 1 showed that the intracellular ADP-ribose concentration could be significantly increased by external ADP-ribose treatment in various solid cancer (brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, and kidney cancer) cells (FIG. 1).

Example 2: Change in Cancer Cell Viability Depending on ADP-Ribose Concentration

Example 2-1: Change in Viability in U-87MG Cells According to Increase in ADP-Ribose Concentration

[0156] 3×10.sup.3 U-87MG cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at final concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM, respectively, and cultured again for 24 hours under conditions of 37° C. and 5% CO.sub.2. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0157] As a result, it was confirmed under a microscope that the rapid growth of brain cancer cells (U-87MG) was observed in the control group not treated with ADP-ribose, whereas the experimental group treated with ADP-ribose showed that the brain cancer cells were inhibited from growing and killed (FIG. 2A). The half-lethal dose and the maximum lethal dose according to the ADP-ribose treatment concentration were quantitatively measured, and as a result, it was confirmed that about half of the brain cancer cells were killed at a concentration of 8 μM, and almost all cells were killed at 32 μM (FIG. 2B).

Example 2-2: Change in Viability in H1975 Cells According to Increase in ADP-Ribose Concentration

[0158] 3×10.sup.3 H1975 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM, respectively, and cultured again for 24 hours under conditions of 37° C. and 5% CO.sub.2. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0159] As a result, it was confirmed under a microscope that the rapid growth of lung cancer cells (H1975) was observed in the control group not treated with ADP-ribose, whereas the experimental group treated with ADP-ribose showed that the lung cancer cells were inhibited from growing and killed (FIG. 3A). The half-lethal dose and the maximum lethal dose according to the ADP-ribose treatment concentration were quantitatively measured, and as a result, it was confirmed that about half of the lung cancer cells were killed at a concentration of 2 μM, and almost all cells were killed at 32 μM (FIG. 3B).

Example 2-3: Change in Viability in AsPC-1 Cells According to Increase in ADP-Ribose Concentration

[0160] 3×10.sup.3 AsPC-1 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM, respectively, and cultured again for 24 hours under conditions of 37° C. and 5% CO.sub.2. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0161] As a result, it was confirmed under a microscope that the rapid growth of pancreatic cancer cells (AsPC-1) was observed in the control group not treated with ADP-ribose, whereas the experimental group treated with ADP-ribose showed that the pancreatic cancer cells were inhibited from growing and killed (FIG. 4A). The half-lethal dose and the maximum lethal dose according to the ADP-ribose treatment concentration were quantitatively measured, and as a result, it was confirmed that about half of the pancreatic cancer cells were killed at a concentration of 2 μM, and almost all cells were killed at 32 μM (FIG. 4B).

Example 2-4: Change in Viability in Hep G2 Cells According to Increase in ADP-Ribose Concentration

[0162] 3×10.sup.3 Hep G2 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM, respectively, and cultured again for 24 hours under conditions of 37° C. and 5% CO.sub.2. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0163] As a result, it was confirmed under a microscope that the rapid growth of liver cancer cells (Hep G2) was observed in the control group not treated with ADP-ribose, whereas the experimental group treated with ADP-ribose showed that the liver cancer cells were inhibited from growing and killed (FIG. 5A). The half-lethal dose and the maximum lethal dose according to the ADP-ribose treatment concentration were quantitatively measured, and as a result, it was confirmed that about half of the liver cancer cells were killed at a concentration of 4 μM, and almost all cells were killed at 32 μM (FIG. 5B).

Example 2-5: Change in Viability in MDA-MB-231 Cells According to Increase in ADP-Ribose Concentration

[0164] 3×10.sup.3 MDA-MB-231 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM, respectively, and cultured again for 24 hours under conditions of 37° C. and 5% CO.sub.2. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0165] As a result, it was confirmed under a microscope that the rapid growth of breast cancer cells (MDA-MB-231) was observed in the control group not treated with ADP-ribose, whereas the experimental group treated with ADP-ribose showed that the breast cancer cells were inhibited from growing and killed (FIG. 6A). The half-lethal dose and the maximum lethal dose according to the ADP-ribose treatment concentration were quantitatively measured, and as a result, it was confirmed that about half of the breast cancer cells were killed at a concentration of 16 μM, and almost all cells were killed at 32 μM (FIG. 6B).

Example 2-6: Change in Viability in HCT116 Cells According to Increase in ADP-Ribose Concentration

[0166] 3×10.sup.3 HCT116 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% Ca.sub.2, treated with ADP-ribose at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM, respectively, and cultured again for 24 hours under conditions of 37° C. and 5% CO.sub.2. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0167] As a result, it was confirmed under a microscope that the rapid growth of colon cancer cells (HCT116) was observed in the control group not treated with ADP-ribose, whereas the experimental group treated with ADP-ribose showed that the colon cancer cells were inhibited from growing and killed (FIG. 7A). The half-lethal dose and the maximum lethal dose according to the ADP-ribose treatment concentration were quantitatively measured, and as a result, it was confirmed that about half of the colon cancer cells were killed at a concentration of 8 μM, and almost all cells were killed at 32 μM (FIG. 7B).

Example 2-7: Change in Viability in Caki-1 Cells According to Increase in ADP-Ribose Concentration

[0168] 3×10.sup.3 Caki-1 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% Ca.sub.2, treated with ADP-ribose at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM, respectively, and cultured again for 24 hours under conditions of 37° C. and 5% CO.sub.2. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0169] As a result, it was confirmed under a microscope that the rapid growth of kidney cancer cells (Caki-1) was observed in the control group not treated with ADP-ribose, whereas the experimental group treated with ADP-ribose showed that the kidney cancer cells were inhibited from growing and killed (FIG. 8A). The half-lethal dose and the maximum lethal dose according to the ADP-ribose treatment concentration were quantitatively measured, and as a result, it was confirmed that about half of the kidney cancer cells were killed at a concentration of 16 μM, and almost all cells were killed at 32 μM (FIG. 8B).

Example 2-8: Change in Viability in SNU-1 or OVCAR-3 Cells According to an Increase in ADP-Ribose Concentration

[0170] 3×10.sup.3 SNU-1 or OVCAR-3 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at concentrations of 32 μM, respectively, and cultured again for 24 hours under conditions of 37° C. and 5% CO.sub.2. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0171] As a result, it was confirmed that when gastric cancer cells (SNU-1) or ovarian cancer cells (OVCAR-3) were treated with ADP-ribose, the cells were killed significantly compared to the control group (FIG. 9).

[0172] Summarizing the above, it was confirmed through the cell experiment of Example 2 that ADP-ribose had an anti-cancer effect on cells of various solid cancers (brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, kidney cancer, gastric cancer, and ovarian cancer) (FIGS. 2 to 9).

Example 3: Change in Metastasis and Invasion Ability of Metastatic Cancer Cells by ADP-Ribose

[0173] When the growth of cancer cells is inhibited and death is induced, the ability of cancer cells to metastasize or invade may also be affected. As in Example 2, the effects of inhibiting the growth of cancer cells and inducing cell death were clearly observed by treatment with ADP-ribose. Thus, it is possible to expect that the treatment with ADP-ribose may induce a reduction in metastasis and invasion ability of cancer cells.

Example 3-1: Change in Metastasis and Invasion Ability of AsPC-1 Cells by ADP-Ribose

[0174] Analysis was performed using a transwell having polycarbonate membrane filters with 8.0 μm pore size. The transwell was put in a 24-well plate, and AsPC-1 cells were seeded on the upper surface of the insert at a density of 3.5×10.sup.5 cells per well, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2. After culturing, the transwell was carefully washed with sterile water and then placed in 100% methanol to fix the attached cells. After fixation, the cells were stained with hematoxylin and an eosin reagent to observe the cells.

[0175] As a result, it was confirmed that invasion and metastasis of pancreatic cancer cells (AsPC-1) from the upper part to the lower part of the transwell were easily observed in the control group, whereas the experimental group treated with 16 μM ADP-ribose showed that the invasion and metastasis of pancreatic cancer cells were significantly inhibited (FIG. 10A). The metastasis and invasion inhibitory effect of ADP-ribose on pancreatic cancer cells were quantitatively measured, and as a result, it was confirmed that the metastasis and invasion ability were significantly reduced (P<0.001) compared to the control group (FIG. 10B).

Example 3-2: Change in Metastasis and Invasion Ability of MDA-MB-231 Cells by ADP-Ribose

[0176] Analysis was performed using a transwell having polycarbonate membrane filters with 8.0 μm pore size. The transwell was put in a 24-well plate, and MDA-MB-231 cells were seeded on the upper surface of the insert at a density of 3.5×10.sup.5 cells per well, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2. After culturing, the transwell was carefully washed with sterile water and then placed in 100% methanol to fix the attached cells. After fixation, the cells were stained with hematoxylin and an eosin reagent to observe the cells.

[0177] As a result, it was confirmed that invasion and metastasis of breast cancer cells (MDA-MB-231) from the upper part to the lower part of the transwell were easily observed in the control group, whereas the experimental group treated with 32 μM ADP-ribose showed that the invasion and metastasis of breast cancer cells were significantly inhibited (FIG. 11A). The metastasis and invasion inhibitory effect of ADP-ribose on breast cancer cells were quantitatively measured, and as a result, it was confirmed that the metastasis and invasion ability were significantly reduced (P<0.001) compared to the control group (FIG. 11B).

[0178] Summarizing the above, it was confirmed in Example 3 that ADP-ribose inhibited the metastasis and invasion ability of solid cancer (pancreatic cancer, breast cancer) cells, thereby providing excellent therapeutic effects on metastatic cancer (FIGS. 10 and 11).

Example 4: Comparison and Verification of Anti-Cancer Efficacy According to the Concentration and Administration Route of ADP-Ribose Using Animal Models

Example 4-1: Change in Tumor Volume in Animal Models Injected Subcutaneously with Various Concentrations of ADP-Ribose

[0179] Pancreatic cancer (AsPC-1) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into four groups: the control group (Control) and groups injected subcutaneously with 2, 20, and 40 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, each concentration of ADP-ribose was subcutaneously injected 3 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0180] As a result of measuring the volume of pancreatic cancer injected over time, the final volume of tumors in the control group after the last dose was about 2155.8 mm.sup.3, and the final tumor volumes of the groups injected subcutaneously with 2, 20, and 40 mg/kg of ADP-ribose were 1015.8 mm.sup.3, 496.6 mm.sup.3, and 336.2 mm.sup.3, respectively. In other words, in all administration groups, tumor volume was reduced statistically significantly (P<0.001) compared to the control group. Specifically, compared to the group injected subcutaneously with 2 mg/kg of ADP-ribose, the group injected with 20 mg/kg of ADP-ribose had a significant reduction (P<0.001) in tumor volume, and the group injected with 40 mg/kg of ADP-ribose subcutaneously had the smallest tumor volume and showed a statistically significant decrease (P<0.001) as compared to the group injected subcutaneously with 2 mg/kg ADP-ribose (FIGS. 12A and 12B).

Example 4-2: Change in Tumor Volume in Animal Models Injected Intravenously with Various Concentrations of ADP-Ribose

[0181] Kidney cancer (Caki-1) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into 4 groups: the control group (Control) and groups injected intravenously with 2, 10, and 20 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, each concentration of ADP-ribose was subcutaneously injected 3 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0182] As a result of measuring the volume of the kidney cancer injected over time, the final volume of tumors in the control group after the last dose was about 2074.9 mm.sup.3, and the final tumor volumes of the groups injected intravenously with 2, 10, and 20 mg/kg of ADP-ribose were 706.7 mm.sup.3, 331.7 mm.sup.3, and 227.2 mm.sup.3, respectively. In other words, in all injection groups, tumor volume was reduced statistically significantly (P<0.001) compared to the control group. Specifically, compared to the group injected intravenously with 2 mg/kg of ADP-ribose, the group injected intravenously with 10 mg/kg of ADP-ribose had a significant reduction (P<0.001) in tumor volume, and the group injected intravenously with 20 mg/kg of ADP-ribose had the smallest tumor volume and showed a statistically significant decrease (P<0.001) as compared to the groups injected intravenously with 2 mg/kg ADP-ribose and with 10 mg/kg ADP-ribose (FIGS. 13A and 13B).

Example 4-3: Change in Tumor Volume in Animal Models Administered Orally with Various Concentrations of ADP-Ribose

[0183] Pancreatic cancer (AsPC-1) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into four groups: the control group (Control) and groups administered orally with 10, 40, and 80 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, each concentration of ADP-ribose was orally administered 5 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0184] As a result of measuring the volume of pancreatic cancer injected over time, the final volume of tumors in the control group (Control) after the last dose was about 3194.3 mm.sup.3, and the final tumor volumes of the groups injected orally with 10, 40, and 80 mg/kg of ADP-ribose were 1798.6 mm.sup.3, 1175.9 mm.sup.3, and 1056.9 mm.sup.3, respectively. In other words, in all oral administration groups, tumor volume was reduced statistically significantly (P<0.001) compared to the control group. Compared to the group administered orally with 10 mg/kg of ADP-ribose, the tumor volume of the groups injected orally with 40 and 80 mg/kg of ADP-ribose was significantly reduced (P<0.001) (FIGS. 14A and 14B).

[0185] Summarizing the above, it was reconfirmed that ADP-ribose had an anti-cancer effect against various solid cancers through the animal experiment of Example 4, which proceeded after Examples 2 and 3 showing the cell experiment results (FIGS. 12 to 14).

Example 5: Change in Cancer Cell Viability by Combination Treatment of ADP-Ribose and Low-Dose Anti-Cancer Drug

Example 5-1: Change in Viability in U-87MG Cells by the Combination Treatment of ADP-Ribose and Low-Dose Bevacizumab

[0186] 3×10.sup.3 Brain cancer (U-87MG) cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at a concentration of 1.6 μM and bevacizumab at 15 mM, individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0187] As a result, it was confirmed that when brain cancer cells were treated with bevacizumab alone, about 68% of all cancer cells survived, but when treated in combination with ADP-ribose, the viability of cancer cells was significantly reduced to about 18% (P<0.001) (FIG. 15A).

Example 5-2: Change in Viability in H1975 Cells by Combination Treatment of ADP-Ribose and Low-Dose Osimertinib

[0188] 3×10.sup.3 Lung cancer (H1975) cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at a concentration of 1.6 μM and osimertinib at 1 nM, individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0189] As a result, it was confirmed that when lung cancer cells were treated with osimertinib alone, about 50% of all cancer cells survived, but when treated in combination with ADP-ribose, the viability of cancer cells was significantly reduced to about 0.9% (P<0.001) (FIG. 15B).

Example 5-3: Change in Viability in AsPC-1 Cells by Combination Treatment of ADP-Ribose and Low-Dose Gemcitabine

[0190] 3×10.sup.3 Pancreatic cancer (AsPC-1) cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at a concentration of 1.6 μM and gemcitabine at 1 μM, individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0191] As a result, it was confirmed that when pancreatic cancer cells were treated with gemcitabine alone, about 54% of all cancer cells survived, but when treated in combination with ADP-ribose, the viability of cancer cells was significantly reduced to about 1% (P<0.001) (FIG. 15C).

Example 5-4: Change in Viability in Hep G2 Cells by Combination Treatment of ADP-Ribose and Low-Dose Sorafenib

[0192] 3×10.sup.3 Liver cancer (Hep G2) cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at a concentration of 1.6 μM and sorafenib at 1 μM, individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0193] As a result, it was confirmed that when liver cancer cells were treated with sorafenib alone, about 49% of all cancer cells survived, but when treated in combination with ADP-ribose, the viability of cancer cells was significantly reduced to about 0.9% (P<0.001) (FIG. 15D).

Example 5-5: Change in Viability in MDA-MB-231 Cells by Combination Treatment of ADP-Ribose and Low-Dose Herceptin

[0194] 3×10.sup.3 Breast cancer (MDA-MB-231) cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at a concentration of 3.2 μM and herceptin at 2.5 μM, individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0195] As a result, it was confirmed that when breast cancer cells were treated with 2.5 μM of herceptin alone, as many as 81% of all cancer cells survived due to herceptin resistance, but when treated in combination with ADP-ribose, the viability of cancer cells was significantly reduced to about 20% (P<0.001) (FIG. 15E).

Example 5-6: Change in Viability in HCT116 Cells by Combination Treatment of ADP-Ribose and Low-Dose 5-Fluorouracil

[0196] 3×10.sup.3 Colon cancer (HCT116) cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with ADP-ribose at a concentration of 1.6 μM/100 μl and 5-fluorouracil (5-FU) at 10 μM, individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0197] As a result, it was confirmed that when colon cancer cells were treated with 10 μM of 5-FU alone, about 51% of all cancer cells survived, but when treated in combination with ADP-ribose, the viability of cancer cells was significantly reduced to about 12% (P<0.001) (FIG. 15F).

[0198] Summarizing the above, it could be appreciated from Example 5 that when administered in combination with ADP-ribose and existing anti-cancer drugs, a synergistic anti-cancer effect on solid cancer was shown, and ADP-ribose could be employed not only as an anti-cancer drug for solid cancer, but also be usefully employed as an anti-cancer adjuvant for existing anti-cancer drug (FIG. 15).

Example 6: Change in Tumor Volume in Animal Models by Combination Administration of ADP-Ribose and Low-Dose Anti-Cancer Drug

Example 6-1: Change in Tumor Volume in Brain Cancer Animal Models by Combination Administration of ADP-Ribose and Low-Dose Bevacizumab

[0199] Brain cancer (U-87MG) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into three groups: the control group (Control), single intraperitoneal administration of 25 mg/kg of Avastin (ingredient name: bevacizumab), and combination administration of Avastin+10 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, Avastin was administered intraperitoneally twice a week, and ADP-ribose at 10 mg/kg was injected subcutaneously 3 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0200] As a result of measuring and comparing the volume of brain cancer over time, the final volume of tumors in the control group (Control) after the last dose was about 2527.1 mm.sup.3, and the final tumor volume of the Avastin-administered group was about 1598.9 mm.sup.3, which was about 37% lower than that of the control group, whereas the final tumor volume of the group after combination treatment of Avastin and ADP-ribose was about 334.5 mm.sup.3, which showed a significant reduction of about 87% (P<0.001) compared to the control group, and even compared to the group treated with Avastin alone, the tumor volume of the combination treatment group was significantly reduced by about 79% (P<0.001) (FIG. 16A).

Example 6-2: Change in Tumor Volume in Lung Cancer Animal Models by Combination Administration of ADP-Ribose and Low-Dose Osimertinib

[0201] Lung cancer (H1975) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into three groups: the control group (Control), single oral administration of 1 mg/kg of osimertinib, and combination administration of osimertinib+10 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, osimertinib was administered orally twice a week, and ADP-ribose at 10 mg/kg was injected subcutaneously 3 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0202] As a result of measuring and comparing the volume of lung cancer over time, the final volume of tumors in the control group (Control) after the last dose was about 2265.5 mm.sup.3, and the final tumor volume of the osimertinib-administered group was about 1881.9 mm.sup.3, which was about 17% lower than that of the control group, whereas the final tumor volume of the group after combination treatment of osimertinib and ADP-ribose was about 424.7 mm.sup.3, which showed a significant reduction of about 81% (P<0.001) compared to the control group, and even compared to the group treated with osimertinib alone, the tumor volume of the combination treatment group was significantly reduced by about 77% (P<0.001) (FIG. 16B).

Example 6-3: Change in Tumor Volume in Pancreatic Cancer Animal Models by Combination Administration of ADP-Ribose and Low-Dose Gemcitabine

[0203] Pancreatic cancer (AsPC-1) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into three groups: the control group (Control), single intraperitoneal administration of 50 mg/kg of gemcitabine, and combination administration of gemcitabine+10 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, gemcitabine was administered intraperitoneally twice a week, and ADP-ribose at 10 mg/kg was injected subcutaneously 3 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0204] As a result of measuring and comparing the volume of pancreatic cancer over time, the final volume of tumors in the control group (Control) after the last dose was about 2174.3 mm.sup.3, and the final tumor volume of the gemcitabine-administered group was about 1815.3 mm.sup.3, which was about 17% lower than that of the control group, whereas the final tumor volume of the group after combination treatment of gemcitabine and ADP-ribose was about 444.3 mm.sup.3, which showed a significant reduction of about 80% (P<0.001) compared to the control group, and even compared to the group treated with gemcitabine alone, the tumor volume of the combination treatment group was significantly reduced by about 76% (P<0.001) (FIG. 16C).

Example 6-4: Change in Tumor Volume in Liver Cancer Animal Models by Combination Administration of ADP-Ribose and Low-Dose Sorafenib

[0205] Liver cancer (Hep G2) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into three groups: the control group (Control), single oral administration of 10 mg/kg of sorafenib, and combination administration of sorafenib+10 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, sorafenib was administered orally 5 times a week, and ADP-ribose at 10 mg/kg was injected subcutaneously 3 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0206] As a result of measuring and comparing the volume of liver cancer over time, the final volume of tumors in the control group after the last dose was about 2322.4 mm.sup.3, and the final tumor volume of the sorafenib-administered group was about 1849.8 mm.sup.3, which was about 20% lower than that of the control group, whereas the final tumor volume of the group after combination treatment of sorafenib and ADP-ribose was about 546 mm.sup.3, which showed a significant reduction of about 76% (P<0.001) compared to the control group, and even compared to the group treated with sorafenib alone, the tumor volume of the combination treatment group was significantly reduced by about 70% (P<0.001) (FIG. 16D).

[0207] Summarizing the above, it could be appreciated from the animal experiment of Example 6, which proceeded after the cell experiment of Example 5, that when administered in combination with ADP-ribose and existing anti-cancer drugs, a synergistic anti-cancer effect on solid cancer was shown even when the anti-cancer drug was administered at a low-dose, and ADP-ribose could be employed not only as an anti-cancer drug for solid cancer, but also be usefully employed as an anti-cancer adjuvant for existing anti-cancer drug (FIG. 16).

Example 7: Change in Cancer Cells of ADP-Ribose by Radiation and ADP-Ribose Treatment

Example 7-1: Change Over Time in ADP-Ribose by Treatment with Various Doses of Radiation and ADP-Ribose in U-87MG Cells

[0208] U-87MG cells were divided into five groups: no treatment group (Control group), groups treated with 1, 2, and 5 Gy of radiation, and ADP-ribose treatment group, and cultured in a 6-well plate for 0, 4, 8, 16, and 24 hours, respectively. Then, the medium was removed, and the supernatant was prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The prepared supernatant was used for ELISA analysis for the detection of ADP-ribose.

[0209] FIG. 17A is a graph showing the intracellular increase (%) in ADP-ribose according to increasing radiation doses of 1, 2, and 5 Gy and 8 μM ADP-ribose treatment in U-87MG cells, compared to the control group. It was confirmed that in the group using increasing radiation doses of 1, 2, and 5 Gy, the amount of ADP-ribose increased to a maximum at 4 hours, then began to decrease from 8 hours, and recovered to almost the same amount as that of the untreated group at 24 hours, but the group treated with ADP-ribose maintained a significant (P<0.001) increase in the amount of ADP-ribose in the cells compared to the control group even after 24 hours (FIG. 17A).

Example 7-2: Change Over Time in ADP-Ribose by Treatment with Various Doses of Radiation and ADP-Ribose in Caki-1 Cells

[0210] Caki-1 cells were divided into five groups: no treatment group (Control group), groups treated with 1, 2, and 5 Gy of radiation, and ADP-ribose treatment group, and cultured in a 6-well plate for 0, 4, 8, 16, and 24 hours, respectively. Then, the medium was removed, and the supernatant was prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The prepared supernatant was used for ELISA analysis for the detection of ADP-ribose.

[0211] FIG. 17B is a graph showing the intracellular increase (%) in ADP-ribose according to increasing radiation doses of 1, 2, and 5 Gy and 16 μM ADP-ribose treatment in Caki-1 cells, compared to the control group. It was confirmed that in the group using increasing radiation doses of 1, 2, and 5 Gy, the amount of ADP-ribose increased to a maximum at 4 hours and recovered to almost the same amount as that of the untreated group from 8 hours, but the group treated with ADP-ribose maintained a significant (P<0.001) increase in the amount of ADP-ribose in the cells compared to the control group even after 24 hours (FIG. 17B).

Example 7-3: Change Over Time in ADP-Ribose by Treatment with Various Doses of Radiation and ADP-Ribose in AsPC-1 Cells

[0212] AsPC-1 cells were divided into five groups: no treatment group (Control group), groups treated with 1, 2, and 5 Gy of radiation, and ADP-ribose treatment group, and cultured in a 6-well plate for 0, 4, 8, 16, and 24 hours, respectively. Then, the medium was removed, and the supernatant was prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The prepared supernatant was used for ELISA analysis for the detection of ADP-ribose.

[0213] FIG. 17C is a graph showing the intracellular increase (%) in ADP-ribose according to increasing radiation doses of 1, 2, and 5 Gy and 2 μM ADP-ribose treatment in AsPC-1 cells, compared to the control group. It was confirmed that in the group using increasing radiation doses of 1, 2, and 5 Gy, the amount of ADP-ribose increased to a maximum at 4 hours and recovered to almost the same amount as that of the untreated group from 16 hours, but the group treated with ADP-ribose maintained a significant (P<0.001) increase in the amount of ADP-ribose in the cells compared to the control group even after 24 hours (FIG. 17C).

Example 7-4: Change Over Time in ADP-Ribose by Treatment with Various Doses of Radiation and ADP-Ribose in MDA-MB-231 Cells

[0214] MDA-MB-231 cells were divided into five groups: no treatment group (Control group), groups treated with 1, 2, and 5 Gy of radiation, and ADP-ribose treatment group, and cultured in a 6-well plate for 0, 4, 8, 16, and 24 hours, respectively. Then, the medium was removed, and the supernatant was prepared by treatment with RIPA buffer and 1% sodium dodecyl sulfate (SDS). The prepared supernatant was used for ELISA analysis for the detection of ADP-ribose.

[0215] FIG. 17D is a graph showing the intracellular increase (%) in ADP-ribose according to increasing radiation doses of 1, 2, and 5 Gy and 16 μM ADP-ribose treatment in MDA-MB-231 cells, compared to the control group. It was confirmed that in the group using increasing radiation doses of 1, 2, and 5 Gy, the amount of ADP-ribose increased to a maximum at 4 hours and recovered to almost the same amount as that of the untreated group from 16 hours, but the group treated with ADP-ribose maintained a significant (P<0.001) increase in the amount of ADP-ribose in the cells compared to the control group even after 24 hours (FIG. 17D).

[0216] Summarizing the above, it was confirmed in Example 7 that external ADP-ribose treatment in various solid cancer cells could maintain the increase in intracellular ADP-ribose concentration above the concentration of ADP-ribose in cancer cells that elevated upon irradiation (FIG. 17). The anti-cancer effect of irradiation could be expected through the death action caused by damage to genetic material, but to overcome this, cancer cells continuously repair DNA strands through a process known as adenosine diphosphate ribosylation. However, as can be appreciated in FIG. 17, if ADP-ribose, which increases only temporarily for the repair of DNA strands destroyed by irradiation, is supported to accumulate continuously, disturbance of biochemical action may be caused, which may be expected to have a synergistic anti-cancer effect of radiation therapy, and furthermore, a possibility of using anti-cancer radiation at a reduced dose to overcome resistance to radiation therapy and reduce side effects.

Example 8: Change in Cancer Cell Viability by Combination Treatment of ADP-Ribose and Radiation

Example 8-1: Change in Viability in U-87MG Cells by Combination Treatment of ADP-Ribose and Tolerable Dose of Radiation

[0217] 3×10.sup.3 Brain cancer (U-87MG) cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with 8 μM of ADP-ribose and 2 or 5 Gy dose of radiation individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0218] As a result, it was confirmed that when U-87MG cells were treated with 2 Gy dose of radiation alone, about 88% of cancer cells showed resistance to survival compared to the untreated group (control), but when treated with ADP-ribose in combination, the survival rate of cancer cells decreased significantly (P<0.001) to about 12.3% even when 2 Gy dose of radiation was used (FIG. 18A).

Example 8-2: Change in Viability in H1975 Cells by Combination Treatment of ADP-Ribose and Tolerable Dose of Radiation

[0219] 3×10.sup.3 H1975 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% Ca.sub.2, treated with 2 μM of ADP-ribose and 2 or 5 Gy dose of radiation individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0220] As a result, it was confirmed that when H1975 cells were treated with 2 Gy dose of radiation alone, about 75% of cancer cells showed resistance to survival compared to the untreated group (Control), but when treated with ADP-ribose in combination, the survival rate of cancer cells decreased significantly (P<0.001) to about 8% even when 2 Gy dose of radiation was used (FIG. 18B).

Example 8-3: Change in Viability in AsPC-1 Cells by Combination Treatment of ADP-Ribose and Tolerable Dose of Radiation

[0221] 3×10.sup.3 AsPC-1 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with 2 μM of ADP-ribose and 2 or 5 Gy dose of radiation individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0222] As a result, it was confirmed that when AsPC-1 cells were treated with 2 Gy dose of radiation alone, about 87.3% of cancer cells showed resistance to survival compared to the untreated group (Control), but when treated with ADP-ribose in combination, the survival rate of cancer cells decreased significantly (P<0.001) to about 8% even when 2 Gy dose of radiation was used (FIG. 18C).

Example 8-4: Change in Viability in Hep G2 Cells by Combination Treatment of ADP-Ribose and Tolerable Dose of Radiation

[0223] 3×10.sup.3 Hep G2 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with 4 μM of ADP-ribose and 2 or 5 Gy dose of radiation individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0224] As a result, it was confirmed that when Hep G2 cells were treated with 2 Gy dose of radiation alone, about 82.3% of cancer cells showed resistance to survival compared to the untreated group (Control), but when treated with ADP-ribose in combination, the survival rate of cancer cells decreased significantly (P<0.001) to about 7.3% even when 2 Gy dose of radiation was used (FIG. 18D).

Example 8-5: Change in Viability in MDA-MB-231 Cells by Combination Treatment of ADP-Ribose and Tolerable Dose of Radiation

[0225] 3×10.sup.3 MDA-MB-231 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with 16 μM of ADP-ribose and 2 or 5 Gy dose of radiation individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0226] As a result, it was confirmed that when MDA-MB-231 cells were treated with 2 Gy dose of radiation alone, about 90.6% of cancer cells showed resistance to survival compared to the untreated group (Control), but when treated with ADP-ribose in combination, the survival rate of cancer cells decreased significantly (P<0.001) to about 11.3% even when 2 Gy dose of radiation was used (FIG. 18E).

Example 8-6: Change in Viability in HCT116 Cells by Combination Treatment of ADP-Ribose and Tolerable Dose of Radiation

[0227] 3×10.sup.3 HCT116 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with 16 μM of ADP-ribose and 2 or 5 Gy dose of radiation individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0228] As a result, it was confirmed that when HCT116 cells were treated with 2 Gy dose of radiation alone, about 90.3% of cancer cells showed resistance to survival compared to the untreated group (Control), but when treated with ADP-ribose in combination, the survival rate of cancer cells decreased significantly (P<0.001) to about 14% even when 2 Gy dose of radiation was used (FIG. 18F).

Example 8-7: Change in Viability in Caki-1 Cells by Combination Treatment of ADP-Ribose and Tolerable Dose of Radiation

[0229] 3×10.sup.3 Caki-1 cells were cultured in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2, treated with 16 μM of ADP-ribose and 2 or 5 Gy dose of radiation individually or in combination, and cultured for 24 hours under conditions of 37° C. and 5% CO.sub.2 again. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After the reaction was completed, the reagent was removed, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm cell viability.

[0230] As a result, it was confirmed that when Caki-1 cells were treated with 2 Gy dose of radiation alone, about 94.7% of cancer cells showed resistance to survival compared to the untreated group (Control), but when treated with ADP-ribose in combination, the survival rate of cancer cells decreased significantly (P<0.001) to about 22.7% even when 2 Gy dose of radiation was used (FIG. 18G).

[0231] Summarizing the above, it could be appreciated from Example 8 that when administered in combination with ADP-ribose and radiation, a synergistic anti-cancer effect on solid cancer was shown, and ADP-ribose could be employed not only as an anti-cancer drug for solid cancer but also as an anti-cancer adjuvant for radiation therapy (FIG. 18).

Example 9: Change in Tumor Volume in Animal Models by Combination Treatment of ADP-Ribose and Tolerable Dose of Radiation

Example 9-1: Change in Tumor Volume in Animal Models of Brain Cancer by the Combination Treatment of ADP-Ribose and Tolerable Dose of Radiation

[0232] Brain cancer (U-87MG) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into three groups: the control group, 2 Gy dose of radiation alone, and combination administration of 2 Gy dose radiation+10 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, 2 Gy dose of radiation was irradiated twice at 1 Gy each, and 10 mg/kg of ADP-ribose was injected subcutaneously 3 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0233] As a result of measuring and comparing the volume of brain cancer over time, FIG. 19A showed that the final volume of tumors in the control group after the last dose was about 2545.2 mm.sup.3, and the final tumor volume of the group irradiated with 2 Gy dose of radiation alone was about 1472.1 mm.sup.3, which was about 42% lower than that of the control group, whereas the final tumor volume of the group after combination treatment of 2 Gy dose of radiation and ADP-ribose was about 725.2 mm.sup.3, which showed a significant reduction of about 71.5% (P<0.001) compared to the control group, and even compared to the group treated with 2 Gy dose of radiation alone, the tumor volume of the combination treatment group was significantly reduced by about 50.7% (P<0.001) (FIG. 19A).

Example 9-2: Change in Tumor Volume in Animal Models of Pancreatic Cancer by Combination Administration of ADP-Ribose and Tolerable Dose of Radiation

[0234] Pancreatic cancer (AsPC-1) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into three groups: the control group (Control), 2 Gy dose of radiation alone, and combination administration of 2 Gy dose radiation+10 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, 2 Gy dose of radiation was irradiated twice at 1 Gy each, and 10 mg/kg of ADP-ribose was injected subcutaneously 3 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0235] As a result of measuring and comparing the volume of pancreatic cancer over time, FIG. 19B showed that the final volume of tumors in the control group (Control) after the last dose was about 2384.3 mm.sup.3, and the final tumor volume of the group irradiated with 2 Gy dose of radiation alone was about 1725.5 mm.sup.3, which was about 27.6% lower than that of the control group, whereas the final tumor volume of the group after combination treatment of 2 Gy dose of radiation and ADP-ribose was about 736.2 mm.sup.3, which showed a significant reduction of about 69.1% (P<0.001) compared to the control group, and even compared to the group treated with 2 Gy dose of radiation alone, the tumor volume of the combination treatment group was significantly reduced by about 57.3% (P<0.001) (FIG. 19B).

Example 9-3: Change in Tumor Volume in Animal Models of Breast Cancer by Combination Administration of ADP-Ribose and Tolerable Dose of Radiation

[0236] Breast cancer (MDA-MB-231) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into three groups: the control group (Control), 2 Gy dose of radiation alone, and combination administration of 2 Gy dose radiation+20 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, 2 Gy dose of radiation was irradiated twice at 1 Gy each, and 20 mg/kg of ADP-ribose was injected subcutaneously 3 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0237] As a result of measuring and comparing the volume of breast cancer over time, FIG. 19C showed that the final volume of tumors in the control group (Control) after the last dose was about 2336.1 mm.sup.3, and the final tumor volume of the group irradiated with 2 Gy dose of radiation alone was about 1459.6 mm.sup.3, which was about 37.5% lower than that of the control group, whereas the final tumor volume of the group after combination treatment of 2 Gy dose of radiation and ADP-ribose was about 640.4 mm.sup.3, which showed a significant reduction of about 72.6% (P<0.001) compared to the control group, and even compared to the group treated with 2 Gy dose of radiation alone, the tumor volume of the combination treatment group was significantly reduced by about 56.1% (P<0.001).

Example 9-4: Change in Tumor Volume in Animal Models of Kidney Cancer Treated with ADP-Ribose and Tolerable Dose of Radiation

[0238] Kidney cancer (Caki-1) cells (1×10.sup.7) were inoculated into 5-week-old BALB/c nude mice on the back flank, and the mice were divided into three groups: the control group (Control), 2 Gy dose of radiation alone, and combination administration of 2 Gy dose radiation+20 mg/kg of ADP-ribose. When the tumors were grown to a volume of approximately 150 mm.sup.3, 2 Gy dose of radiation was irradiated twice at 1 Gy each, and 20 mg/kg of ADP-ribose was injected subcutaneously 3 times a week. The tumor size was measured using a digital caliper, and the results of changes in tumor volume were compared by the group.

[0239] As a result of measuring and comparing the volume of kidney cancer over time, FIG. 19D showed that the final volume of tumors in the control group (Control) after the last dose was about 2620.5 mm.sup.3, and the final tumor volume of the group irradiated with 2 Gy dose of radiation alone was about 2454.5 mm.sup.3, which was about 6.3% lower than that of the control group, whereas the final tumor volume of the group after combination treatment of 2 Gy dose of radiation and ADP-ribose was about 786.4 mm.sup.3, which showed a significant reduction of about 70% (P<0.001) compared to the control group, and even compared to the group treated with 2 Gy dose of radiation alone, the tumor volume of the combination treatment group was significantly reduced by about 68% (P<0.001) (FIG. 19D).

[0240] Summarizing the above, it could be appreciated from the animal experiment of Example 9, which proceeded after the cell experiment of Example 8, that when administered in combination with ADP-ribose and radiation, a synergistic anti-cancer effect on solid cancer was shown, and ADP-ribose could be employed not only as an anti-cancer drug for solid cancer but also as an anti-cancer adjuvant for radiation therapy (FIG. 19).

Example 10: Confirmation of Toxicity in Normal Cells when Treated with ADP-Ribose

[0241] In the case of an anti-cancer drug, it is important to exhibit toxicity specifically to cancer cells without exhibiting toxicity to normal cells. Therefore, whether ADP-ribose exhibited toxicity to normal cells according to the present disclosure was evaluated.

[0242] As normal cells, human colon fibroblast (CCD-18Co) and human dermal papilla cell (HDPC) cells were used. First, the respective normal cells (5×10.sup.3) were cultured in DMEM medium in a 96-well plate for 24 hours under conditions of 37° C. and 5% CO.sub.2. Then, the cells were divided into a total of 4 groups: ADP-ribose untreated group (Untreated), and treated groups (treated with increased concentrations of 25, 50, 100 ul). Cell viability was compared by MTT assay after 24, 48, and 72 hours of drug treatment.

[0243] As a result, it was confirmed that no toxicity was observed when ADP-ribose was treated in all normal cells (FIGS. 20A and 20B: CCD-18Co, and FIGS. 20C and 20D: HDPC). Therefore, together with the experimental results of the Examples above, the present Example shows that ADP-ribose is capable of acting specifically on cancer cells to be usefully employed as an anti-cancer drug.