ANTICANCER FLUORESCENT SUBSTANCE DERIVED FROM NATURAL MATERIALS

Abstract

Provided is a composition for preventing or treating cancer, the composition including resveratrone, resveratrone glucoside, or a combination thereof as an active ingredient.

Claims

1-13. (canceled)

14. A method for preventing or treating cancer, the method comprising administering resveratrone, resveratrone glucoside, or a combination thereof to a subject in need thereof.

15. A method of claim 14, wherein the cancer is selected from the group consisting of solid cancer, primary cancer, metastatic cancer, and recurrent cancer.

16. A method of claim 14, wherein the method further includes measuring fluorescence intensity in the subject.

17. A method of claim 14, wherein the method is for detecting or diagnosing cancer.

18. A pharmaceutical composition for preventing or treating cancer, the composition comprising resveratrone, resveratrone glucoside, or a combination thereof as an active ingredient.

19. The composition of claim 18, wherein the composition further comprises radioisotopes, quantum dots, MM contrast agents, or diagnostic antibodies.

20. The composition of claim 18, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

21. A contrast agent composition comprising resveratrone, resveratrone glucoside, or a combination thereof as an active ingredient.

22. The contrast agent composition of claim 21, wherein the composition is for specifically detecting cancer.

23. The contrast agent composition of claim 21, wherein the composition further includes radioisotopes, quantum dots, MM contrast agents, or diagnostic antibodies.

24. The contrast agent composition of claim 21, wherein the composition is administered to a living body or a sample.

25. A method for detecting the presence of cancer cells, the method comprising: administering resveratrone, resveratrone glucoside, or a combination thereof to a subject; and measuring fluorescence intensity in the subject.

26. The method of claim 25, wherein the subject is a mammal or a human.

27. The method of claim 25, wherein the subject is cells cultured in vitro, tissue isolated from a body, an organoid, or a combination thereof.

28. A method for screening for a cancer therapeutic drug, the method comprising: contacting resveratrone, resveratrone glucoside, or a combination thereof with cancer cells; culturing the cancer cells; and measuring fluorescence intensity in the cancer cell culture.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0061] FIG. 1A is an illustration showing anticancer effect and cancer cell detection ability of resveratrone and resveratrone glucoside, and FIG. 1B shows excitation and emission wavelength ranges of resveratrone and resveratrone glucoside;

[0062] FIG. 2A shows fractions of cells showing apoptosis after treatment of normal cells and cancer cells with resveratrone, and FIG. 2B shows FACS graphs showing fractions of cells showing apoptosis after treatment with resveratrone glucoside, in which the right section of each graph represents an apoptosis zone;

[0063] FIG. 3A and FIG. 3B show graphs of TUNEL analysis results of observing apoptotic signals after treatment with resveratrone and resveratrone glucoside, respectively;

[0064] FIG. 4 shows graphs of Western blotting results of examining apoptosis after treatment of normal breast cells and breast cancer cells with resveratrone;

[0065] FIG. 5 shows optical microscope images (DIC) showing cell morphology after treatment of normal breast cells and breast cancer cells with resveratrone;

[0066] FIG. 6A shows fluorescence microscope images showing fluorescence intensity after treatment of normal breast cells and breast cancer cells with resveratrone glucoside; FIG. 6B shows a graph of measuring and comparing fluorescence intensity after treatment of normal breast cells and breast cancer cells with resveratrone glucoside;

[0067] FIG. 7 shows a fluorescence microscope image showing HeLa cells treated with resveratrone;

[0068] FIG. 8A and FIG. 8B show optical microscope (DIC) and two photon fluorescence microscope images which were observed over time after treatment of Zebrafish embryos with resveratrone and resveratrone glucoside, respectively; and

[0069] FIG. 9 shows optical microscope (DIC) and fluorescence microscope images of observing apoptotic effects after treatment of organoids with resveratrone.

MODE OF DISCLOSURE

[0070] Hereinafter, the present disclosure will be described in detail with reference to the examples. However, the following examples are only for illustrating the present disclosure, and the content of the present disclosure is not limited by the following examples.

Example 1: Measurement of Cancer Cell Apoptosis by Resveratrone and Resveratrone Glucoside

[0071] 1-1. Measurement by Fluorescence-Activated Cell Sorting (FACS)

[0072] Cancer cell apoptotic effects of resveratrone and resveratrone glucoside (see U.S. Pat. No. 9,708,23762) were measured by FACS using Annexin-V binding to phosphatidiylserine (PS) which exists in the inner leaflet of the cytoplasmic membrane and is exposed to the outer leaflet of cell membrane during apoptosis.

[0073] In detail, apoptosis of normal cells or cancer cells, each treated with resveratrone or resveratrone glucoside, was evaluated by measuring signals of Alexa dye-conjugated annexin V using FACS. HeLa, MCF7, SW480, SW620, and HCC1954 which are cancer cells, and NIH3T3 and MCF10A which are normal cells, each treated with 50 μM resveratrone or 100 μM resveratrone glucoside, were cultured under conditions of 37° C. and 5% CO.sub.2 for 24 hours. Thereafter, media was removed, and the cells were washed with PBS buffer twice to remove remaining resveratrone or resveratrone glucoside. The cells were collected and centrifuged to remove supernatants. Annexin-bound buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl.sub.2, pH 7.4) was added and resuspended. 5 μl of Alexa Fluor 647-conjugated annexin V was added to the resuspended cells at the cell volume of 100 μl, followed by incubation for 15 minutes. 1 ml of binding buffer was added to the incubated cells, followed by centrifugation. The supernatant was removed to remove remaining Alexa Fluor 647-conjugated annexin V. Thereafter, 500 μl of annexin-binding buffer was further added, and cells were passed through a cell strainer cap and stored in a round-bottom tube. The number of apoptotic cells in each 1000 cells of normal cells and cancer cells was counted by repeated FACS analysis using an excitation wavelength of 633 nm, at which Alexa Fluor 647 emits fluorescence, but resveratrone does not emit fluorescence (FIGS. 2A and 2B).

[0074] As a result, it was confirmed that, as compared with that of normal cells, the number of apoptotic cells in the cancer cells was increased to at least 15 times to about 40 times when treated with resveratrone, and increased to about 22 times when treated with resveratrone glucoside (Table 1).

TABLE-US-00001 TABLE 1 resveratrone resveratrone glucoside Kind of cell normal cancer normal cell cancer cell cell cell NIH3T3 MCF10A HeLa MCF7 SW480 SW620 HCC1954 MCF10A MCF7 Apoptotic 2.0 1.0 20.0 26.0 15.5 17.4 39.3 0.7 15.5 fraction (%)

[0075] 1-2. Measurement by TUNEL Assay Cancer cell apoptotic effects of resveratrone and resveratrone glucoside, which were evaluated in Example 1-1, were re-evaluated by TUNEL assay.

[0076] In detail, a cancer cell MCF7 and a normal cell MCF10A were treated with 50 μM of resveratrone or resveratrone glucoside, respectively, and incubated for 24 hours. Thereafter, the cells were treated with 0.5% PBS-Triton X-100 and incubated for 10 minutes to increase cell permeability. The cells were treated with terminal deoxynucleotide transferase and tetra-methyl-rhodamine dUTP, followed by incubation at 37° C. for 1 hour. Next, the cells were washed with PBS three times, and treated with DAPI dye for nuclear counterstaining. Apoptotic signals were observed using fluorescence images.

[0077] As a result, TUNEL detecting apoptotic signals was observed only in the cancer cell MCF7, indicating that resveratrone or resveratrone glucoside had no effect on normal cells and exhibited selective apoptotic ability against cancer cells (FIGS. 3A and 3B).

Example 2: Measurement of Cancer Cell Apoptosis Induction of Resveratrone

[0078] 2-1. Measurement by Western Blotting

[0079] Cancer cell apoptotic effects of resveratrone and resveratrone glucoside, which were evaluated in Examples 1-1 and 1-2, were re-evaluated by Western blotting.

[0080] In detail, a cancer cell MCF7 and a normal cell MCF10A were treated with 50 μM of resveratrone, respectively, and incubated for 48 hours. The incubated cells were harvested and lysed with a NETN buffer (150 mM NaCl, 20 mM Tris/Cl pH 8.0, 0.5% v/v NP-40, 1 mM EDTA) containing a protease inhibitor, and then conjugated with a cleaved parp antibody, and sequentially, with a secondary antibody. Then, the presence or absence thereof was examined.

[0081] As a result, Cleaved parp which was not observed in the resveratrone-treated normal cells was observed in the resveratrone-treated cancer cells, indicating that resveratrone induced apoptosis of cancer cells (FIG. 4).

[0082] 2-2. Measurement by Optical Microscopy

[0083] Cancer cell apoptotic effects of resveratrone and resveratrone glucoside, which were evaluated in the above Examples, were examined by observing morphology.

[0084] In detail, a normal cell MCF10A and a cancer cell MCF7 were treated with 50 μM of resveratrone, respectively, and incubated for 48 hours. After incubation, media was removed, and the cells were washed with PBS buffer three times. Changes of cell morphology in PBS buffer were observed and compared by optical microscopy.

[0085] As a result, cell morphology of the normal cell MCF10A was clearly observed irrespective of treatment with resveratrone, whereas the resveratrone-treated cancer cell MCF7 showed morphological features of apoptosis, as compared with the control group (FIG. 5).

Example 3. Evaluation of Function of Resveratrone and Resveratrone Glucoside as Contrast Agent

[0086] 3-1. Resveratrone Glucoside

[0087] Function of resveratrone glucoside as a contrast agent was evaluated.

[0088] In detail, a cancer cell MCF7 and a normal cell MCF10A were treated with 300 μM of resveratrone glucoside, respectively, and incubated for 3 hours. Then, they were washed with PBS buffer twice, and cells were resuspended in PBS buffer, respectively. Imaging of each cell suspension was performed by confocal fluorescence microscopy using 800 nm pulse laser as a light source and a two-photon absorption phenomenon of resveratrone, and mean fluorescence intensity of resveratrone glucoside in the normal cell and cancer cell was measured and compared.

[0089] As a result of the experiment, the cancer cell MCF7 showed stronger fluorescence intensity than the normal cell MCF10A (FIG. 6A), and the intensity was about 170% stronger (FIG. 6B). Therefore, it is suggested that resveratrone glucoside may be used to diagnose cancer cells by distinguishing cancer cells from normal cells.

[0090] 3-2. Resveratrone

[0091] Function of resveratrone as a contrast agent was evaluated.

[0092] In detail, media was removed from HeLa cell culture. The cells were washed with PBS buffer three times, and treated with MeOH at 20° C., and fixed by incubation for 5 minutes. Thereafter, the cells were treated with 30 uM of resveratrone and incubated for 4 hours. Then, the cells were washed with PBS buffer twice and treated with Mowiol which is an imaging medium. The Mowiol-treated HeLa cells were excited at 405 nm, and a fluorescence image of resveratrone was examined.

[0093] As a result, it was confirmed that resveratrone may also function as a contrast agent (FIG. 7).

[0094] 3-3. Evaluation of Cytotoxicity of Resveratrone as Contrast Agent

[0095] It was examined whether resveratrone exhibited cytotoxicity when used as a contrast agent.

[0096] In detail, zebrafish embryos (donated by Hyunsook Lee Lab, School of Biological Sciences, Seoul National University) were cultured, and treated with 300 μM of resveratrone or resveratrone glucoside. At 2 hr, 20 hr, 48 hr, and 72 hr after treatment, their changes were observed.

[0097] As a result, resveratrone induced no cytotoxicity during development of zebrafish embryos, indicating no toxicity as a contrast agent (FIG. 8A). Resveratrone glucoside also induced no cytotoxicity during development of zebrafish embryos, but weak fluorescence was observed, as compared with resveratrone (FIG. 8B).

Example 4. Examination of Resveratrone Effect in Organoids

[0098] As an alternative to animal experiments, it was confirmed that resveratrone had a significant cancer cell apoptotic effect on organoids.

[0099] In detail, as an alternative to a pancreatic tissue of a wild-type mouse and an individual with cancer, a pancreatic tissue having G12D mutation of Kras gene was obtained from a transgenic mouse, and the pancreatic tissue was dissociated with a dissociation solution, filtered. Then, a cell pellet containing ductal cells was collected by centrifugation, and incubated with a biological substrate material. The dissociation solution containing Hank's Balanced Salt Solution (HBSS) (3 ml), collagenase P (5 mg/ml), and DNase 1 (1 Units/μl) was used in a volume of 3 ml per pancreatic tissue (Vpan=about 1.08 mg/mm.sup.3). When the collected cell pellet was incubated with the biological substrate material, three-dimensional pancreatic organoids were prepared by incubation under conditions of 37° C. and 5% CO.sub.2 with Matrigel which is a gelatinous protein mixture secreted from the murine Engelbreth-Holm-Swarm (EHS) sarcoma. Resveratrone and resveratrone glucoside were treated using DMSO as a solvent to reach a specific concentration with respect to the culture. After 72 hr, the medium was replaced by a fresh culture medium, and the above treatment was repeated. 12 hr after final treatment, changes of the organoids were recorded using an inverted fluorescence microscope (available from Zeiss) capable of photographing 450 mm fluorescence. To examine morphology of the organoids, differential interference contrast (DIC) images were also photographed.

[0100] As a result, unlike mouse pancreatic organoids, organoids having G12D mutation of Kras gene showed apoptosis induction in response to resveratrone (FIG. 9). Accordingly, it is predicted that the effects identical or similar to those demonstrated in the present Example may also be obtained in vivo.

[0101] As described above, the present disclosure has been described with reference to the examples. It will be understood by those skilled in the art to which the present disclosure pertains that the present disclosure may be embodied in modified forms without departing from essential characteristics thereof. Therefore, the disclosed embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the present disclosure is indicated by the appended claims rather than by the foregoing description. It shall be interpreted that all differences within the equivalent scope are included in the present disclosure.