KR101114438B1 - A Composition for treating or preventing breast cancer comprising Rhapontigenin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a lapontigenin (3,3 ', 5-trihydroxy-4'-methoxystilbene) compound and a pharmaceutically acceptable carrier, adjuvant, or vehicle. It relates to a composition for preventing or treating breast cancer comprising the compound as an active ingredient.

Lapontigenin, Cancer, Breast Cancer, Treatment

Description

A composition for treating or preventing breast cancer comprising Rhapontigenin}

The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a lapontigenin (3,3 ', 5-trihydroxy-4'-methoxystilbene) compound and a pharmaceutically acceptable carrier, adjuvant, or vehicle. It relates to a composition for preventing or treating breast cancer comprising the compound as an active ingredient.

In general, breast cancer is the second most common cancer in the world after lung cancer, and the fifth-highest cancer death rate. According to statistics from the Korea Central Cancer Registry released in 2008, breast cancer ranks sixth among all cancers and ranks first among female cancers.

It is urgent to develop drugs with fewer side effects for the treatment of such breast cancer. We implemented the present invention to develop a drug having a low side effect while fully fulfilling its role as an anticancer agent.

Intracellular responses to general metabolic stress are apoptosis, and in cancer cells these apoptosis is important for inhibiting the activity of cancer cells. In recent years, the relationship between apoptosis and autophagy has been gradually revealed (Self-eating and self-killing: crosstalk between autophagy and apoptosis.M. Chiara Maiuri et al., Septamber 2007 volume 8 741-752)

Autophagy is the catabolism of catabolism through the incorporation, cleavage and regeneration of proteins, organelles and cytoplasm, in response to deficiencies or stress in cells to maintain cell homeostasis. Autophagy also works to eliminate pathogens or cells that have killed apoptosis. Although autophagy is known to play a role in cell survival, paradoxically, autophagy, which is not inhibited, is involved in apoptosis resulting from gradual intracellular consumption . (Role of autophagy in cancer.Nat Rev Cancer. 2007 Dec; 7 (12): 961-7)

The most likely assumption is that degradation via autophagy primarily assists cell survival, but in cells with metabolic imbalances, consumption by autophagy is faster than the ability to synthesize them, leading to cell death.

Stilbenes are primarily secondary by-products that are present in the leaves, bark and wood parts of higher plants and are biologically active in the form of derivatives such as resveratrol in plants. Stilbene is also made in response to damage or colony formation by microbial pathogens, which show a high accumulation of stilbene derivatives during the invasion of various types of fungi, particularly the interaction between colony- and living tissues. One can speculate that these play an important potential role in resisting death in plants.

Rhubarb has been used as a traditional medicine in Asia such as Korea, Japan and China. In particular, rapontin extracted from the root of rhubarb is known to have anti-allergic and melanin inhibitory effects. Rapontin is a stilbene-based substance, and the effects of anti-allergic, anti-lipid, and anti-inflammatory effects have been reported for rapontigenin, which has increased bioactivity through the conversion of rapontin.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a compound for treating or preventing breast cancer that is safe and effective for the human body.

In order to achieve the above object, the present invention provides a composition comprising a lafontigenin (3,3 ', 5-trihydroxy-4'-methoxystilbene) compound and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

In one embodiment of the present invention, the compound is preferably a rhubarb-derived compound, but is not limited thereto.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a lafontigenin (3,3 ', 5-trihydroxy-4'-methoxystilbene) compound and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

In a preferred embodiment of the present invention, the cancer is preferably breast cancer, but is not limited thereto.

In one embodiment of the invention, the composition preferably further comprises a chloroquine (chloroquine) that is an autophagy inhibitor, but is not limited thereto.

FR122047 of the present invention is a selective inhibitor of COX-1, IC50 values for inhibition of human COX-1 and COX-2 are 0.028 and 65 μM, respectively (Ochi, T., Motoyama, Y., Goto, T. The analgesic effect profile of FR122047, a selective cyclooxygenase-1 inhibitor, in chemical nociceptive models.Eur J Pharmacol 391 49-54 (2000)).

In another embodiment of the present invention, the composition preferably further includes PD98059, an inhibitor of ERK (Extracellular signal-regulated kinases; hereinafter referred to as 'ERK') pathway inhibitor, but is not limited thereto.

The ERK-MAP kinase used in the present invention is a serine / threonine kinase that is commonly present in eukaryotes, and it is various life phenomena in response to various extracellular stimuli (cell proliferation, neuronal differentiation, cytokinetic activity, egg cell maturation, etc.). It plays a central role in the derivation of. In other words, ERK and MAP kinases play a really central role in the intracellular signal transduction system, which is caused by the specific binding of various cell proliferation and differentiation factors to each receptor present on the target cell surface.

In the intracellular signal transduction system (ERK-MAP kinase cascade), it is known that several signal molecules function upstream of ERK-MAP kinase, and Ras, Raf-1, and MEK are best interpreted among them. Etc. Specifically, Ras, a type of GTP binding protein, is activated in response to an external stimulus, and then Raf-1 (serine / threonine kinase), which is a type of MAP kinase kinase kinase (MAPKKK), is activated. Raf-1 then phosphorylates / activates MEK1 / 2, a type of MAP kinase kinase that has a specific substrate specificity that phosphorylates both serine / threonine residues and tyrosine residues. Activated MEK1 / 2 here represents an inactive MAP kinase (ERK1 / 2), T (threonine) and Y in the Thr-Glu-Tyr (TEY) array present between its kinase subdomains (Ⅶ and Ⅷ). Phosphorylation of (tyrosine) residues converts them to active MAP kinases. The activated MAP kinase (ERK1 / 2) thus migrates from the cytoplasm into the nucleus and induces phosphorylation / activation of several transcription factors, thereby leading to the expression of the final physiological response.

It is known that the ERK MAP kinase cascade (path) is involved in the expression of various physiological responses as described above, and in particular it has been found to play an essential role in promoting cell proliferation. In other words, it is expected to inhibit cell proliferation by blocking the ERK-MAP kinase cascade.

PD 98059 (2′-amino-3´-methoxyflavone) of the present invention is a potent, cell-permeable and selective inhibitor of MAPK / ERK kinase 1 (MAP kinase 1 or MEK1), and inhibits the activation of MEK1, It inhibits subsequent phosphorylation and activation of kinase. Its IC50 value is in the range of 1-20 μM.

Purified compositions contemplated for use herein include purified extract portions having the properties described herein from all sources, whether in natural or variant form of plants or species, whether natural, synthetic or recombinant.

The invention also provides for the use of synthetic formulations having the properties of the compositions of the invention. Such synthetic preparations may be prepared based on the chemical structure and / or functionality of the compositions of the invention described above. Also provided are analogs, homologs, and pseudomers having the chemical structure and functionality of the compositions of the present invention.

As used herein, "analogues, homologs, and pseudologues" of the compositions of the present invention are those of the present invention wherein one or more residues are not added and / or substituted and / or removed from a compound that is not clearly structurally similar. Compounds that differ from the structure of the composition. However, in all cases these compounds have substantially the same activity as the compositions of the present invention. Thus, "analogue" refers to a compound that has the same basic structure as the composition of the invention but differs in several residues, and "homolog" is a compound that differs from the composition of the invention by the addition and / or removal and / or substitution of a limited number of residues. "Phyto" means a compound that does not have intrinsic structural similarity to the composition of the present invention. Such compounds will exhibit the bioactive properties of the compositions of the present invention.

Rapontigenin (a) used in the present invention and PD98059 (b), a chlorokin and / or ERK pathway inhibitor, which are additionally included autophagy inhibitors, are independently of combination partners (a) and (b) or By means of the use of different fixed compositions with distinct amounts of combination partners (a) and (b), that is to say that they can be administered simultaneously or at different time points. That is, the components of the composition may be administered simultaneously or staggered in time, ie at the same time or at different time intervals at different times for any of the components.

Very preferably, the time interval is selected such that the effect on the treated disease in the use of the combination of components is greater than the effect obtained by the use of only one of the combination partners (a) and (b). The ratio of the total amount of the composition partner (a) to the combination partner (b) to be administered in the combination formulation may vary depending, for example, on the needs of the patient subset to be treated or the needs of a single patient. It may be due to a specific disease, age, sex, weight, etc. of the patient. Preferably, the mutual amplification of one or more beneficial effects, for example the effects of the combination partners (a) and (b), in particular the effect beyond the synergistic, eg additive effect, further advantageous effects, Less side effects, associated therapeutic effects at non-effective doses of either or both of the composition partners (a) and (b), and very preferably the strong synergy of the composition partners (a) and (b) exist.

Reference to the composition partners (a) and (b) of the present invention is also understood to mean including pharmaceutically acceptable salts. If the composition partners (a) and (b) have, for example, one or more base centers, they can form acid addition salts. In addition, corresponding acid addition salts may be formed, if necessary, to have additional base centers present. Combination partners (a) and (b) with acid groups (eg COOH) may also form salts with bases. The composition partner (a) or (b) or a pharmaceutically acceptable salt thereof may be used in the form of a hydrate or may include other solvents used for crystallization.

As used herein, the term "anticancer" is the broadest meaning encompassing all of the prevention, treatment, alleviation and alleviation of symptoms.

In addition, the term "cancer prevention" in this specification is the broadest meaning including inhibiting any one or more of the initiation stage, promotion stage, progression stage and metastasis stage of cancer. Among them, it refers to inhibiting cancer development at the onset and promotion stage of cancer. It also includes preventing cancer through a blocking mechanism that inhibits the carcinogen from reacting with or reaching the target within the cell. It also includes preventing cancer through a suppressing mechanism that inhibits the processes in which cells exposed to carcinogens appear as neoplasia. It also includes preventing cancer by facilitating ex vivo release of carcinogenic or precarcinogenic substances. It also includes metabolizing precancerous substances to prevent cancer.

The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compositions of the present invention may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

In various embodiments of the invention, the anticancer agent that can be used together is a chemotherapeutic agent, radiotherapy agent, or both. In certain embodiments, the chemotherapeutic agent can be an alkylating agent, an anti metabolism, a hormone or an antagonist, an antibody, or a mixture thereof. Chemotherapeutic agents are, for example, pemetrexed, gemcitabine, cisplatin, methotrexate,

Remetrexate, or a mixture thereof. Examples of alkylating agents include nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan or chlorambucil. Other examples of chemotherapeutic agents include nitrosoureas such as carmustine, romustine or semustine; Ethyleneimines and / or methyl-melamines such as triethylenemelamine, triethylene thiophosphoramide or hexamethylmelamine; Alkyl sulfonates such as busulfan; And triazines such as dacarbazine. Other anticancer agents include anti-metabolites such as folic acid analogs (eg methotrexate, trimetrexate or pemetrexed); Pyrimidine analogs (eg 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside, 5-azacytidine or 2,2'-difluorodeoxycytidine); Purine analogs (eg, 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycofomycin, erythrohydroxynonyladenine, fludarabine salt

Or 2-chlorodeoxyadenosine); And / or type I topoisomerase inhibitors (eg, camptothecin, topotecan or irinotecan). In some embodiments, the anticancer agent is a natural product, such as epipodophyllotoxin (eg, etoposide or teniposide); And / or from vinca alkaloids (eg vinblastine, vincristine or vinorelbine)

Can be derived. In certain embodiments, the anticancer agent may be an antibiotic such as actinomycin D, doxorubicin or bleomycin. In various embodiments, the anticancer agent can be a radiosensitizer, such as 5-bromodeoxyuridine, 5-iododeoxyuridine or bromodeoxycytidine. In some embodiments, the anticancer agent may be a platinum coordination complex such as cisplatin, carboplatin or oxaliplatin. The anticancer agent may be hydroxyurea and / or methylhydrazine derivatives such as N-methylhydrazine or procarbazine. In some embodiments, the radiotherapy can be gamma-radiation, X-ray radiation, ultraviolet radiation, visible radiation, infrared radiation, and microwave radiation.

The pharmaceutical composition according to the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, ointments, creams, external preparations, suppositories, and sterile injectable solutions. It may be used in formulated in any form suitable for pharmaceutical formulations.

In the compositions according to the present invention, the preferred dosage of the compound of the present invention depends on the subject's age, sex, weight, symptoms, degree of disease, drug form, route of administration and duration, but may be appropriately selected by those skilled in the art. have. However, for the desired effect, the composition of the present invention is preferably administered at 0.001 to 10 g / kg body weight per day. Administration may be administered once a day or may be divided several times. In addition, the dosage may be increased or decreased depending on age, sex, weight, degree of disease, route of administration, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes, such as parenteral, oral, and all modes of administration can be expected, for example, oral, rectal Or by intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

On the other hand, the composition according to the present invention, there is no serious toxicity and side effects can be used with confidence even for long-term use for the purpose of prevention.

On the other hand, in the case of formulating a compound of the present invention into tablets, capsules, chewing tablets, small bags, granules, powders, liquid solutions, suspensions, dispersions, emulsions, syrups and the like for the purpose of oral administration, gum arabic, Corn starch, binders such as microcrystalline cellulose or gelatin, excipients such as dicalcium phosphate or lactose, disintegrants such as alginic acid, corn starch or potato starch, lubricants such as magnesium stearate, sweeteners such as sucrose or saccharin and Flavoring agents such as peppermint, methyl salicylate or fruit flavors may be included, and when the dosage unit form is a capsule, liquid carriers such as polyethylene glycol or fatty oil may be included in addition to the above components.

It is contemplated that the invention described herein is not limited to the particular methods, protocols, and reagents described as being varied. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention in any way.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, the preferred methods, devices, and materials are described below.

In the practice of the present invention, many conventional techniques of molecular biology and cell biology are used. These techniques are well known and are described, for example, in Current Protocols in Molecular Biology, Volumes I, II and III, 1997 (F. M. Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.

As can be seen through the above-mentioned means and the following examples, the lapontigenin of the present invention did not induce apoptosis in normal cells, but it can be confirmed that it specifically induces apoptosis in breast cancer cell lines. It can be used as a candidate for the treatment or prevention of cancer later.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

The reagents used in the present invention and where to buy them are as follows;

Rapontin (3,3 ', 5-trihydroxy-4'-methoxystilbene 3-Od-glucoside), Rapontigenin (3,3', 5-trihydroxy-4'-methoxystilbene), Resveratrol (3,4 ', 5) Stilbene-based plant-derived materials, such as -trihydroxy-trans-stilbene), oxyresveratrol (2,3 ', 4,5'-tetrahydroxystilbene), and mulberryside A, were provided by Professor Kim Jung-keun of the Korea Polytechnic University. Distilled phospahate-buffered saline (DPBS), Fatal bovine serum (FBS), Trysin-EDTA, Trypan blue, and DMSO (dimethyl sulfoxide) were purchased from GIBCO. PD98059, N-acetylcysteine (NAC) was purchased from BioMall. Chlorokin, rapamycin, doxorubicin, etoposide and Monodansylcadaverine (MDC) were purchased from Sigma and FR122047 from Calbiochem. Sulforapane was purchased from LKT Laboratories with 2 ', 7'-dichlorodihydrofluorescein diac-etate (DCFH-DA) from Molecular Probes. The reagents were all dissolved in DMSO, and DMSO was used at a concentration of 0.1% for the control of all experiments.

Methanol was added to the rhubarb root and stirred for 48 hours. After standing still, the methanol layer was separated, and methanol was added again and stirred. After standing still again, the methanol layer was separated, distilled, and dried in vacuo to make rapontin (3,3 ', 5-trihydroxy-4'-methoxystilbene 3-Od-glucoside). Pectinase ( pectin hydrolase ) was added to rapontin extracted from rhubarb and reacted with vacuum to produce rapontigenin (3,3 ', 5-trihydroxy-4'-methoxystilbene) (Fig. 1A).

Example  1: Cell culture environment of the present invention

The MCF-7 breast cancer cell line and SKBR3, keratin forming cells (keratinocytes) of HaCaT cells in Dulbecco's modified Eagle's medium (DMEM ), breast cancer cell line MDA-MB 231, MDA MB 468 , T47D cells 37 ℃ using RPMI 1640 medium Incubated in the incubator. Breast normal cell line MCF-10A was cultured in DMEM F / 12. All cultures were purchased from Gibco. All cells were cultured by adding 10% fetal bovine serum (HyClone) to the culture.

Example  2: cells Survival rate  Measure

Cells were incubated in a 96 well plate at a density of 1 × 10 μs and then changed to a culture medium without FBS after 20 to 24 hours, and then treated with a reagent. After 24 or 48 hours, 10 ul of sodium 3 '-[1- (phenylaminocar-bonyl) -3,4-tetrazolium] -bis (4-methoxy-6-nitro) benzene sul-fonic acid hydrate (XTT) After treatment with each well (EzCytox) in a 37 ℃ incubator for 1 hour, the value was measured at 450mm wavelength using an ELISA.

Example  3: Flow cell  analysis( FACS )

The cells were incubated in a 60 mm plate at a density of 4 × 10 μs, and after 20 to 24 hours, the cells were changed to a medium without FBS, and treated with a reagent. After 48 hours, the cells were centrifuged at 1,200 rpm together with the culture medium for 2 minutes, washed with PBS, stained for 20 minutes with a propidium iodide (PI) staining reagent, and the cell cycle change was measured by FACS.

Example  4: Weston Blasting

Cells were incubated in a 100 mm plate at a density of 6 × 10 μs, and after 20-24 hours, the cells were changed to a culture medium without FBS and the reagents were treated. After 24 hours or 48 hours, the cells were collected and washed with PBS, followed by centrifugation, followed by cytolysis reagent (1% Triton X-100, 100 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride) were added for 30 minutes. After centrifugation at 13,000 rpm for 30 minutes, only the supernatant was extracted and protein quantified using Bradford Protein Assay Reagent (Pierce).

Quantified proteins were separated on 10-15% SDS-PAGE gel, transferred to nitrocellulose membranes (0.2 mm, Schleicher and Schuell), and then blocked with 5% skim milk at room temperature for 1 hour, followed by 4 ° C of primary antibody. After reacting with a secondary antibody for 12 hours at room temperature for 1 hour and 30 minutes, it was developed using an enhanced chemiluminescence (ECL) detection kit (Amersham Bioscience).

Primary antibodies (Actin, poly (ADP-ribose) polymerase (PARP), Caspase 9, Caspase 8, ERK1 / 2, phospho-ERK, p38, phospho-p38, JNK, phospho-JNK, LC3) It was purchased from Cell Signaling. Each antibody was diluted 1: 1000 in 5% skim milk.

Example  5: microscopy

MDC staining was incubated at a density of 3 X 10⁴ cells in a 35mm plate, and after 20 to 24 hours to change the culture medium without FBS was added to the reagents. After 36 hours, the MDC staining reagent was reacted in a 37 ° C. incubator for 20 minutes after 10 ul treatment, and then observed using a fluorescence microscope.

Electron microscopic analysis was performed after fixing the cells with 2.5% glutaraldehyde.

Example  6: Casceptin  B activity measurement

After 12 hours of treatment with the drug, the cells were placed in a cell lysis reagent and left on ice for 30 minutes. The supernatant obtained by centrifugation was added together with the active buffer solution and the substrate in a 96-well plate, followed by 30 minutes in a 37 ℃ incubator. And the value was measured using a fluorescent ELISA.

The result of the above embodiment is as follows.

(1) Rapontin and Lafontigenin

Quantitative analysis of rapontin and lapontigenin made by the above experimental method was performed by HPLC (Fig. 1-B).

(2) Confirmation of survival rate of breast cancer cell lines when treating various anticancer substances

The present inventors confirmed cell death effects in breast cancer cell lines MCF-7, MDA-MB 231, MDA-MB 468, T47D, SKBR3, and the like compared to various stilbene-based and anti-cancer-proven substances (FIG. 2, and 3).

As a result, it was confirmed that lapontigenin had a higher cancer cell death effect than oxyresveratrol, which is known to have a superior cell death effect of breast cancer cells. Rapontin had almost no effect on MCF-7 cells, although it showed higher effect than structurally similar anticancer effect factor resveratrol. Compared with substances that have proven anti-cancer effects, the effects of rapontigenin in all five cell lines were excellent. As the cancer cell inhibitory effect of rapontigenin was confirmed in various breast cancer cell lines, several additional experiments were conducted to prove the effect as a therapeutic agent.

(3) Determination of the effect of lafontigenin on normal cell lines

La Ponti genin is in HaCaT cells in la Ponti processes the genin in the same concentration and time of apoptotic breast normal cell line MCF 10A, and human keratin forming cells (keratinocytes) to ensure that only specific to the cause of cell death of breast cancer cell lines The sub-G1 level was measured.

As a result, it was confirmed that lapontigenin did not induce apoptosis in normal cells, but specifically induced apoptosis in breast cancer cell lines (Fig. 4-A).

(4) Confirmation of Apoptosis-Induced Apoptosis in MCF-7 Cells of Lafontigenin

Previous experiments have shown that lapontigenin reduces the survival of breast cancer cell lines. To determine whether this decrease in cell viability is caused by apoptosis, the sub-G1 levels were checked.

The results of increasing apoptosis were obtained in the same manner as the survival test results (Fig. 4-B). In addition, we could observe the fragments of PARP, a marker of apoptosis, using Western blotting experiments, as well as the increased activity of caspase-8 and caspase-9 (Fig. 4-C).

(5) Measurement of the relationship between cell death and ROS by lafontigenin

Since apoptosis has been reported to be caused by the change of intracellular reactive oxygen species, the level of intracellular reactive oxygen species was measured by using rapontigenin in MCF-7 and MCF-10A cell lines. .

As a result, it was observed that the treatment of rapontin in both cell lines resulted in the reduction of reactive oxygen species (Fig. 5-A). In addition, NAC (N-acetyl-cysteine), a well-known antioxidant, was treated together with MCF-7 cells. The experiments carried out did not show a significant change (Fig. 5-B). Sub-G1 was confirmed after treatment with lapontigenin and NAC at the same time, and it was confirmed that there was little ability to inhibit apoptosis by antioxidants (Fig. 5-C). It is thought that rapontigenin actually plays a role as an antioxidant in cells, but it has no relation to the apoptosis effect in breast cancer cell line and the antioxidant effect of this substance.

(6) Confirmation of MAP Kinase Pathway Regulation of Lafontigenin in MCF-7 Cell Line

We investigated whether apoptosis induced by lapontigenin treatment is related to MAP kinase pathway. Other studies have shown that the cancer cell suppression mechanism of lafontigenin causes apoptosis in association with cytochrome P1A1 on the mitochondrial membrane. It has also been reported that cytochrome P1A1 causes apoptosis in association with MAP kinase. Therefore, the activation of major MAP kinase was confirmed after the treatment with lapontigenin over time in MCF-7 cells and normal cell lines.

As a result, activation of ERK was increased only in MCF-7 cells. In contrast, normal cell lines showed little change in activation of ERK. On the other hand, activation of p38 MAPK and JNK was not prominent by lapontigenin treatment. (FIG. 6-A) Inhibitor of MEK pathway to investigate whether breast cancer cell death by lapontigenin treatment is related to ERK pathway. It was confirmed that the apoptosis of MCF-7 cells was reduced when PD98059 was used to inhibit the activation of ERK using PD98059 as the inhibitor. As a result, when PD98059 was treated together, the sub-G1 level was decreased by about 40% compared to the treatment with only lapontigenin alone (Fig. 6-B). These results suggest that the ERK signal pathway in intracellular signal transduction may be related to the apoptosis of breast cancer cell lines by lapontigenin.

(7) Confirmation of autophagy induction and association with apoptosis in MCF-7 cells by lapontigenin treatment

Promoting autophagy by inhibiting mTOR can suppress cancer, and chlorokines have been reported to inhibit tumor growth in mice by inducing autophagy inhibition. These reports confirmed whether autophagy was induced in MCF-7 cell line upon treatment with lafontigenin.

When autophagy occurs, autophagy can be confirmed by accumulation of LC3-II, which is known as a marker of autophagy. In the present invention, LC3-II accumulation tended to increase markedly only in the MCF-7 cell line treated with lapontigenin. , No change was seen in normal cell lines (MCF-10A, HaCaT) (Figs. 7-A, B). These results indicate that lapontigenin induces autophagy as well as apoptosis in MCF-7 cells. As a result of confirming that autophagy was stained using an MDC staining reagent which can be identified when the cells were induced in cells to obtain more clear results, it was confirmed that autophagy was induced in MCF-7 cells by the treatment of lapontigenin (FIG. 7). -C).

Next, pretreatment with chlorokines known to inhibit autophagy and rapamycin known to induce autophagy showed that when apoptosis was inhibited by pretreatment with chlorokines, apoptosis was compared with lapontigenin alone. When the cell death was increased significantly (by nearly 6 times increased apoptosis), and when treated with an autophagy inducer, apoptosis by lapontigenin was reduced (Fig. 7-D). Thus, autophagy induced by lafontigenin is highly related to apoptosis of MCF-7 cells, and autophagy can be expected to have cytoprotection function that inhibits apoptosis. These results suggest that lapontigenin selectively induces cell death in the MCF-7 breast cancer cell line, and that apoptosis involving ERK and especially cytoprotective autophagy plays an important role in the cell death. It is thought that it showed the development potential as a treatment for breast cancer.

(8) Effect on apoptosis in MCF-7 cell line by treatment with another autophagy inducer FR122047.

Several studies have demonstrated the association between autophagy and apoptosis by treating FR122047, which is known to inhibit COX-1.

After treatment with FR122047, the formation of autophagy was confirmed by electron microscopy, MDC staining, and LC3-GFP transduction (Figs. 8-A, B, C). The accumulation of LC3-II was shown to increase (Fig. 8-D, E). Interestingly, the treatment with FR122047 after the pretreatment with chlorokin, an autophagy inhibitor, showed a significant increase in apoptosis, as was the case with the treatment with lapontigenin. It is thought that autophagy-inducing agents such as FR122047 and lapontigenin may increase apoptosis in the breast cancer cell lines when autophagy is inhibited.

(9) Confirmation of autophagy induction and association with apoptosis in MCF-7 cell line by sulforaphane treatment.

Lafontigenin and FR122047 are both drugs that induce apoptosis and autophagy. Sulfonaphane, which is known to inhibit cancer cell growth and induce autophagy, is also used in various breast cancer cell lines based on the fact that autophagy induces apoptosis. Treatment was performed to determine cell viability.

In all five cell lines, cell viability was decreased by 24 and 48 hours treatment (Figs. 9-A, B, C, D, and E), and sulforaphane was treated in MCF-7, a breast cancer cell line. The result of increasing sub-G1 was confirmed (FIG. 10-A) and Western blotting was performed to confirm the increase of LC3-II. Furthermore, after treatment with doxorubicin and etoposide, which is used as an anticancer agent, it was compared with the case of sulforaphane (Fig. 10-B).

As a result, it was found that autophagy was hardly induced when doxorubicin or etoposide was treated to cells. It is thought that doxorubicin and etoposide induce apoptosis with little induction of autphagy. In addition, as a result of confirming the activity of casceptin B, another autophagy confirmation test technique, when treated with sulforaphane and lapontigenin, the activity was similarly high, which may be expected to be higher when co-treated with an autophagy inhibitor. It is judged that (FIG. 10-C).

(10) Schematic diagram of induction of autophagy and apoptosis in breast cancer cell lines by lapontigenin, FR122047, and sulforaphane treatment.

All previous studies provide evidence that apoptosis and autophagy-inducing mechanisms in the breast cancer cell lines of lafontigenin, FR122047 and sulforaphane may occur through similar patterns of signaling rather than through completely different signaling.

Therefore, we try to improve the understanding of the mechanism of action in breast cancer cell lines by drawing a schematic diagram. Lapontigenin, FR122047 and sulforaphane further increased apoptosis by chlorokin's autophagy inhibitory action, and doxorubicin and etoposide were found to cause little autophagy (FIG. 11).

Figure 1 is used in the present invention, the production of lapontin (3,3`, 5-trihydroxy-4`-methoxystilbene 3-Od-glucoside) and lafontigenin (3,3`, 5-trihydroxy-4`-methoxystilbene) Process (A). And analysis result (B) using HPLC;

Figure 2 shows the stilbene-based materials (top) used in the present invention and those (anti-cancer) demonstrated anti-cancer effect (bottom);

3 is a diagram showing the cell viability of various substances in the breast cancer cell lines MCF-7, MDA-MB 231, MDA-MB 468, T47D, SKBR3 by MTT;

Figure 4 is a comparison of Sub-G1 levels of lafontigenin in MCF-7 cells and normal cell lines (MCF-10A, HaCat) (A). Comparison of Sub-G1 Levels of Rapontin and Lafontigenin in MCF-7 Cells (B). Fig. Confirms the activity of PARP and Caspase using Western blotting in MCF-7 cells (C);

5 is a measure of ROS change by the treatment of lafontigenin. ROS measurement by Rapontigenin treatment in MCF-7 cells and MCF-10A cell line (A). Determination of ROS change by treatment of lafontigenin with N-acetyl-cysteine (B). Treated with lapontigenin and NAC (N-acetyl-cysteine), followed by Sub-G1 measurement (C);

Figure 6 confirms the correlation between apoptosis and MAP kinase pathway that occurs during the treatment of lafontigenin in MCF-7 cell line. The activation of the major MAP kinase pathway factors, ERK, p38 MAPK and JNK, was confirmed by Western blotting (A). PD98059, an inhibitor of ERK, was co-treated with lafontigenin to measure Sub-G1 levels using FACS (B);

Figure 7 confirms the autophagy induction by the treatment of lapontigenin in MCF-7 cells. Western blotting was used to confirm the accumulation of LC3-II (A) and to compare the increase of LC3 II with other normal cell lines at different times (B), and to treat MCF-7 cells with lapontigenin. After staining using the MDC staining reagent was confirmed (C), pre-treatment with chlorokines known to inhibit autophagy and rapamycin that activates autophagy to confirm sub-G1 levels (D);

FIG. 8 shows that FR122047 was treated with MCF-7 cells to induce autophagy, and the formation of autophagosome in the cells was confirmed by electron microscopy (A). FR122047 treatment increased the spotting of LC3-GFP (B) and was confirmed to be stained using the MDC staining reagent (C). In FR122047 treatment, the cleavage of PARP was confirmed (D), and the increase of LC3-II by time treatment was confirmed (E). Sub-G1 levels were confirmed by pretreatment of chlorokines known to inhibit autophagy and rapamycin that activates autophagy (F);

9 is treated with sulforaphane to various breast cancer cell lines to confirm cell viability. Breast cancer cell lines MCF-7, MDA-MB 231, MDA-MB 468, T47D, and SKBR3 were treated with sulforaphane by concentration and time, and cell viability was measured using MTT experimental technique;

Figure 10 is treated with sulforaphane MCF-7 cell line confirmed apoptosis and autophagy induction. Sulforapane was treated to determine Sub-G1 levels using FACS (A). Western blotting compared LC3-II with other anticancer agents (B), and measured the activity of casceptin B, confirming the induction of autophagy by the drug and its activity decreased when co-treated with inhibitor (C). Will;

Figure 11 is a diagram showing the induction of autophagy and apoptosis by various anticancer drugs treatment.

Claims (10)

delete delete A pharmaceutical composition for preventing or treating cancer, which comprises a lapontigenin (3,3 ', 5-trihydroxy-4'-methoxystilbene) compound and chlorokin, an autophagy inhibitor. delete 4. The pharmaceutical composition for treating or preventing cancer according to claim 3, wherein the cancer is breast cancer. delete delete delete delete delete

Images