KR20170022868A - B3 Composition for treating cancer containing [1-9-NC]-linusorb B3 - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, containing [1-9-NC]-linusorb B3 (LOB3) derived from linseed as an active ingredient. According to the present invention, LOB3 is derived from natural materials, thereby being less toxic to the human body, and inducing death of cancer cells not by necrocytosis but by apoptosis. Accordingly, LOB3 can be used as a medicine which is safe without side effects such as inflammation or the like, and has excellent anticancer effects.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for treating cancer containing cyclic linseed oil B3. [Composition for treating cancer comprising [1-9-N? C] -linusorb B3}

The present invention relates to a pharmaceutical composition for treating or preventing cancer, which comprises as an active ingredient a cyclic leucine-derived B3 ([1-9-N? C] -linusorb B3) derived from flaxseed.

Chemotherapy to treat cancer can induce apoptosis (also called " apoptosis ") of cancer cells to cause cancer cells to be removed. In cancer cells, once apoptosis is caused, the cancer cells are destroyed and converted to apoptotic bodies, and then the apoptic body is cleared by surrounding macrophages, so that it does not involve side effects such as inflammatory reactions . Therefore, apoptosis induction of cancer cells is known as an effective mechanism for removing malignant tumor cells by treatment with anticancer agents.

Apoptosis is not only a basic cellular activity applied to maintain physiological balance, but also known as a protective mechanism against cancer through the removal of damaged or abnormal cells. The process is driven by the tight regulation of many pro- and anti-apoptotic molecules, and it has been shown that phosphorylation and dephosphorylation of key regulatory proteins are two important cellular mechanisms in terms of cell growth and apoptosis lost.

STAT3 (Signal Transducer and Activator of Transcription 3) is a transcription factor that is activated by phosphorylation by various regulatory factors including Src, which is excessively activated (phosphorylated) in various types of human tumors including blood cancers and solid tumors, Activated STAT3 promotes the expression of target genes such as Bcl-XL, c-myc, and cyclin D1, which are associated with the survival, proliferation and growth of cancer cells, thereby promoting the cancer cell transformation of mutated cells. Src, also known as a cancer-inducible tyrosine kinase, phosphorylates STAT3 tyrosine 705 and transforms STAT3-dependent cells. It has been reported that STAT3 705 tyrosine is phosphorylated in tissues of hematologic, lymphoma, breast, and brain tumor patients.

On the other hand, flaxseed (Linum usitatissimum L.), a plant belonging to the family Linaceae, is spread to Europe through Asia and Canada, and is cultivated only in a cold region above 55 degrees north. Seeds, proteins and substances that can not be found in other seeds, such as essential fatty acids such as omega-3 and omega-6 and lignans, and substances that reduce the incidence of chronic diseases such as coronary arteries and heart disease It is contained in large quantity.

However, immunological studies of peptides isolated from flaxseed were only reported to induce apoptosis and inhibition of T cell proliferation in lung epithelial cancer cells, beginning with suppression of cholate absorption in hepatocytes in 1986. ([1-9-N? C] -linusorb B3 (LOB3), isolated from flaxseed, contains seven hydrophobic amino acids (two isoleucine, two leucine, one valine and two phenyl Alarine) and two special amino acids (proline). As of now, the precise mechanism of action on anticancer efficacy has not been elucidated.

The inventors of the present invention have conducted intensive research to find herbal medicine ingredients having excellent anticancer effect and fewer side effects than known anticancer drugs. As a result, LOB3 derived from natural products inhibited the activity of oncogenes in various carcinomes including glioma, The present inventors have confirmed that they exhibit excellent anticancer effects without side effects of inflammation, and have completed the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition / health functional food composition for preventing or treating cancer, which contains LOB3 as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a pharmaceutical composition for treating or preventing cancer, which comprises cyclic leucovin B3 ([1-9-N? C] -linusorb B3) as an active ingredient.

Further, the present invention provides a health functional food composition for preventing or ameliorating cancer comprising cyclic leucovin B3 ([1-9-N? C] -linusorb B3) as an active ingredient.

The present invention also provides a method for treating or preventing cancer by administering to a subject a composition containing a cyclic leucovin B3 ([1-9-N? C] -linusorb B3) as an active ingredient.

Further, the present invention provides a cancer prevention or therapeutic use of a composition containing cyclic leucovin B3 ([1-9-N? C] -linusorb B3) as an active ingredient.

In one embodiment of the present invention, the cyclic leucovin B3 induces apoptosis of cancer cells.

In another embodiment of the present invention, the cyclic leucovin B3 is characterized by reducing the activity of the cancer inducing proteins Src and STAT3.

In another embodiment of the present invention, the cyclic leucovorin B3 increases the activity of caspase-3 protein in cancer cells and induces apoptosis.

In another embodiment of the present invention, the cyclic leucovorin B3 is characterized by inhibiting the expression of Bcl2 and p53.

In another embodiment of the present invention, the cyclic leucovorin B3 is characterized in that it is derived from flaxseed.

In another embodiment of the present invention, the cancer is characterized by being a glia species.

According to the present invention, since LOB3 is derived from a natural product called flaxseed, it is advantageous in that it is less toxic to humans and is safe.

In addition, according to the present invention, since cancer cell death induced by LOB3 is caused by apoptosis rather than necrosis, side effects such as inflammation can be solved.

Further, according to the present invention, since the anticancer effect is excellent for various carcinomas including glioma, it can be usefully used as a next generation anticancer drug.

Figure 1 shows the chemical structure of LOB3.
Figure 2 shows the results of MTT assays to assess the cytotoxicity of LOB3 to C6 cells.
Figure 3 shows the results of MTT assays to assess cytotoxicity of LOB3 to MDA-MB-231 cells.
Figure 4 shows the results of MTT assays to assess the cytotoxicity of LOB3 to U251 cells.
Fig. 5 shows the results of confirming the cell division inhibition effect of C6 cells by LOB3.
FIG. 6 shows the morphological changes of cell morphology by optical microscope in order to morphologically confirm the cell death effect in C6 cells by LOB3.
Fig. 7 shows the results of Annexin V / PI staining assay to determine whether apoptosis-induced apoptosis of C6 cells was caused by LOB3.
FIG. 8 is a result of confirming nuclear contraction by fluorescence microscopy to confirm that the cell death effect of LOB3 on C6 cells is due to apoptosis.
FIG. 9 shows the results of confirming Actin disruption by fluorescence microscopy to confirm whether the cell death effect of LOB3 on C6 cells is due to apoptosis.
Fig. 10 shows the results of confirming mRNA expression of Bcl-2 and p53 by LOB3 in C6 cells.
FIG. 11 shows the results of confirming the activity of inducing the activity of caspase-3 protein by LOB3 in C6 cells.
Fig. 12 shows the results of confirming the amount of protein expression of p-Src and p-STAT3 by LOB3 in C6 cells.
Fig. 13 shows the results of migration assay of LOB3 in C6 cells.

The present invention provides a pharmaceutical composition for the treatment or prevention of cancer, which comprises as an active ingredient a cyclic leucovin B3 ([1-9-NαC] -linusorb B3) (hereinafter referred to as "LOB3") as an active ingredient.

The LOB3 of the present invention is a cyclic lipid soluble nonapeptide consisting of 7 hydrophobic amino acids (2 isoleucine, 2 leucine, 1 valine and 2 phenylalanine) and 2 prolines, Respectively.

In addition, there is no limitation on the carcinoma that can be prevented, ameliorated or treated by the composition according to the present invention, but it may include glioma, lung cancer, prostate cancer, colon cancer, breast cancer and the like.

The therapeutic compositions of the present invention may further comprise components such as conventional therapeutically active ingredients, other adjuvants, pharmaceutically acceptable carriers, and the like. The pharmaceutically acceptable carrier includes saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and the like.

The term "individual" as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals.

The term "pharmaceutically effective amount" as used herein refers to the type and severity of the disease to be administered, the age and sex of the patient, sensitivity to the drug, administration time, administration route and rate of release, Can be readily determined by those skilled in the art in an amount that is determined by factors well known in the medical arts, and can be maximized without adverse effects, taking into account all of the above factors.

The composition of the present invention is not limited as long as it can reach the target tissues. For example, oral administration, arterial injection, intravenous injection, percutaneous injection, intranasal administration, transbronchial administration, or intramuscular administration. The daily dose is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and is preferably administered once a day or divided into several times a day.

The composition of the present invention can be used variously for medicines, foods and beverages for prevention and improvement of cancer diseases, and can be used in powder, granule, tablet, capsule or beverage form.

In the present invention, cytotoxicity of C6 (rat glioma cell) cancer cells was evaluated in order to examine the anticancer efficacy of LOB3, and the cancer cell killing effect was confirmed.

In order to understand the principle of cancer cell death in LOB3, LOB3 was treated with LOB3 to observe changes in cell morphology. As a result, it was found that apoptotic blebbing and apoptotic body, which are specific for apoptosis, Was formed.

Further, in order to further confirm whether the cancer cell killing effect of LOB3 is due to apoptosis, annexin V / PI staining for detecting cell suicide marker annexin V and PI which is a cell necrosis marker after treating C6 cell with LOB3 As a result, it was observed that the number of annexin V-stained cells (annexin V + / PI-) was not increased but PI was not stained.

The annexin V / PI staining method, which is an apoptosis assay, uses phosphatidic serine (PS), which is present in the cell membrane when the cell is killed, to protrude outward and then combine with PS and Annexin V. PI (propidium iodide) is a substance that binds to the nucleus. In the case of necrosis, in which the cell is not apoptosis but is rapidly killed by external shock, there is no PS exposure of the cell membrane. Therefore, Annexin V negative, PI positive appear. That is, annexin V + / PI- means death by apoptosis and annexin V- / PI + means death by cell necrosis.

Thus, cell death by LOB3 means that it is in the form of apoptosis which does not cause any side effects such as inflammation. Unlike apoptosis, necrosis is a phenomenon in which cells and intracellular organelles are exploded by substances that can harm normal cells, causing intracellular substances to migrate out and thereby cause an inflammatory response, whereas apoptosis causes cells to burst Instead, small apoptic bodies surrounded by blebbing and cell membranes are formed and they are phagocytosed by the surrounding cells, so there is no inflammation.

Further, in the present invention, C6 cells were treated with LOB3, stained with DNA, and the nuclear structure of the cells was confirmed. As a result, chromosome condensation, which is a characteristic feature of apoptosis, and generation of nuclear fraction were observed. Through this, we concretely confirmed that the apoptotic effect of LOB3 is induced by inducing apoptosis.

In the present invention, to examine the principle of cancer cell suicide by LOB3, C6 cells were treated with LOB3 to confirm the activity of intracellular oncogene protein. As a result, the expression of the protein was increased in active-caspase 3 and active-caspase 9, which are apoptosis markers of induction of apoptosis, and the activity of Src kinase and its substrate STAT3 was decreased As a result, it was confirmed that LOB3 inhibited the activity of Src kinase, which is a carcinogen, to induce apoptosis.

Furthermore, in the present invention, RT-PCR was carried out in order to confirm the effect of LOB3 on cytokine mRNA expression. As a result, it was confirmed that the degree of mRNA expression of Bcl-2 and p53 was decreased, and it was confirmed that the effect of inducing apoptosis was dependent on the concentration of LOB3.

In addition, in the present invention, migration of cells in C6 cells by LOB3 was inhibited. Therefore, it can be expected that LOB3 effectively inhibits cancer cell metastasis.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1: Cell culture and reagents

C6, MDA-MB-231 (breast cancer cells) and U251 (glioblastoma) cells were cultured in RPMI1640 medium containing DMEM medium or penicillin (100 IU / ml) and streptomycin And cultured in a 100 mm cell culture dish at a density of 70-80%.

LOB3 was purchased from Prairie Tide Chemicals and purchased from [19-NαC] -CLA or Cyclolinopeptide A (CAS No. 33302-55-5).

Example  2: cancer cells Survival rate (cell viability) evaluation

In order to examine the anticancer efficacy of LOB3, toxicity in C6 cells was evaluated by MTT assay.

After Specifically, the handle 1 × 10 ⁶ cell / ml C6 cells plated, and the LOB3 of a 96-well plate at different concentrations (0, 2.5, 5, 10 , 20, 30, 40 μM) CO 2 incubator For 48 hours at 37 ° C. Then, 10 μl of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphinyltetrazolium bromide) solution (stock concentration: 5 mg / ml) was added and additional reaction was induced for 3 hours. For reaction termination and formazan crystal dissolution, 100 μl MTT stop solution (10% SDS in 0.01 M HCl) was added to each well. The cell viability is calculated from the OD value obtained by measuring the absorbance at 570 nm of the amount of MTT reduced to formazan.

In addition, proliferation assay was performed to determine cell viability.

As a result, as shown in FIG. 2, most of the C6 cells were observed to be killed in the LOB3 concentration range of 30-40 uM. As shown in FIG. 3, MDA-MB- Death was observed. Also, as shown in FIG. 4, it was observed that most of U251 cells were also killed at 10 to 20 μM.

Furthermore, as shown in Fig. 5, it was confirmed that cell division by LOB3 was inhibited in C6 cells. Therefore, the possibility of anticancer efficacy of LOB3 was confirmed.

Example  3: Observation of morphological changes of cells

Based on the results of Example 2, in order to investigate the principle of cancer cell death of LOB3, C6 cells were treated with LOB3 and then observed for changes in cell morphology.

Specifically, C6 cells were cultured in a 24-well plate at a concentration of 5 × 10 ⁴ cells / ml, treated with LOB3 at a concentration of 10, 20 and 30 μM, And the morphology was observed using an optical microscope.

As a result, as shown in FIG. 6, from the 24th treatment, blebbing and formation of apoptotic bodies, which are specific for apoptosis, were confirmed (see a yellow arrow).

Example  4: Cancer cells apoptosis  observe

Based on the results of Example 3, it was further proved that cancer cell death was caused by apoptosis using Annexin V, an apoptosis marker, and PI (propidium iodide) staining, a necrosis marker.

Specifically, C6 cells were cultured in a 12-well plate at a concentration of 5 × 10 ⁵ cells / ml, LOB3 was treated at a concentration of 10 and 30 μM, cells were harvested after 24 hours, and centrifuged Only the cell mass was isolated. Then, the cells were suspended in 1 × FACS buffer, treated with Annexin V and PI stain for 15 minutes, and then fluorescence was observed using a flow cytometer. Staurosporin (2.5 μM) was used as a comparative experimental group.

As a result, as shown in Fig. 7, it was confirmed that the number of annexin V cells stained but not stained with PI (annexin V + / PI-) was found to be cancer cell death by apoptosis rather than cell necrosis .

Example  5: Using fluorescence staining Nuclear structure  Confirm change

Based on the results of Example 4, the following experiment was conducted to further confirm whether chromosome condensation, which is a phenomenon specific to apoptosis, and nuclear fraction were observed.

More specifically, C6 cells were cultured in a 12-well plate at a concentration of 2.5 × 10 ⁶ cells / ml, treated with LOB3 at concentrations of 2.5, 25, and 40 μM, fluorescently stained with a confocal microscope Were observed.

As a result, as shown in FIG. 8 and FIG. 9, it was confirmed that when LOB3 was treated, nuclear contraction (see yellow arrow in FIG. 8) and actin disruption, which are characteristic of apoptosis, occurred. In addition, as shown in Fig. 9, it can be confirmed that the expression amount of Caspase3, which is a cell death marker, is increased.

Example  6: LOB3  Of cytotoxic protein mRNA  Identification of effects on expression

In order to confirm the effect of LOB3 on the mRNA expression of apoptosis-suppressing protein in C6 cells, the following experiment was performed using RT-PCR.

To measure the degree of mRNA expression at the transcription level, each sample was treated for a period of time and total RNA was extracted using TRIzol reagent. The extracted total RNA was prepared by using a first strand cDNA synthesis kit (Thermo scientific), and then the same amount of cDNA was amplified by PCR. At this time, the sense and antisense primer sequences of the target proteins used were prepared with reference to the existing literature, and β-actin was used as a control gene (see Table 1). PCR amplification was carried out by using a Hipi PCR kit (Elpis biotech). The sense and antisense primers and the control β-actin primers of the cDNA and target proteins of each experimental group were reacted with 250 μM dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, And 20 μl of Hipi PCR kit containing 1.5 mM of NgCl ₂ . The PCR was performed under the conditions of denaturation at 95 ° C for 45 seconds, annealing at 55 ° C for 45 seconds, and extension at 72 ° C for 1 minute, and a total of 30 cycles were performed. The DNA amplified by PCR was electrophoresed on 1.5% agarose gel, and the intensity of the fractionated DNA bands was measured. The results are shown in FIG.

primer Sequences (5 '-> 3') Bcl2
Forward CAC CCC TGG CAT CTT CTC CTT (SEQ ID NO: 1) Reverse CAC AAT CCT CCC CCA GTT CAC C (SEQ ID NO: 2) p53
Forward CTC TGT CAT CTT CCG TCC CTT C (SEQ ID NO: 3) Reverse AGG ACA GGC ACA AAC ACG AAC (SEQ ID NO: 4) β-actin
Forward GTT ACC AGG GCT GCC TTC TC (SEQ ID NO: 5) Reverse GAT GGT GAT GGG TTT CCC GT (SEQ ID NO: 6)

As a result, as shown in Fig. 10, it was confirmed that the degree of mRNA expression of Bcl-2 and p53, which are pro-apoptosis markers, was decreased by LOB3 in C6 cells. Therefore, it can be seen that LOB3 has a strong cell apoptosis inducing ability in a concentration-dependent manner.

Example  7: Identification of target proteins and signaling pathways

In order to analyze the specific molecular mechanism of cancer cell suicide by LOB3, the activity of intracellular oncogene protein was confirmed by Western blotting.

Specifically, C6 cells, a Rattus glioma cell line cultured using DMEM medium containing penicillin (100 IU / ml) and streptomycin (100 μg / ml) and 5% FBS were cultured at a density of 7 × 10 ⁶ cells / ml Lt; / RTI > dish. Afterwards, each fraction was treated and after a certain time according to the drug, the cells were collected and the cells were disrupted using a lysis buffer and a sonicator to obtain a western sample. The protein concentration of each sample was measured with BSA as a standard, and SDS-PAGE was performed with each sample amount as the protein concentration based on the thus obtained value, and the proteins were blotted on a PVDF membrane using a Western blotting method The membrane was blocked with 5% non-fat dried milk (Bio-rad), and the target protein antibody and signaling protein antibody (pro-caspase 3,9 / cleaved caspase 3,9 / Bcl-2 / , p-Stat3, β-actin), and the secondary antibody solution is treated and washed again after the washing step. Then, ECL solution (Amersham, England) was evenly distributed on the membrane in the dark room and sensitized with an X-ray film. The results are shown in FIGS. 11 and 12.

As a result, as shown in FIG. 11, the increase in the amount of protein expression was confirmed in active-caspase 3 and active-caspase 9, which are apoptosis markers of apoptosis inducing factors, and as shown in FIG. 12, (P-Src, p-STAT3) proteins of Src kinase and its substrate STAT3 were decreased, confirming that LOB3 induces apoptosis by inhibiting the activity of oncogenes.

Example  8: Confirmation of cell mobility inhibition

In order to confirm that cell migration was inhibited by LOB3, the following experiment was carried out.

C6 cells were cultured on a 12-well plate at a concentration of 1 × 10 ⁶ cells / ml, and then a gap was formed between the cells and cells using a physical method. Then, LOB3 was treated at different concentrations , 24 and 30 h) were taken using an optical microscope. In addition, a numerical value is shown using a data calculation program.

As a result, as shown in Fig. 13, it was confirmed that when LOB3 was treated, cell migration was suppressed in C6 cells.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> RESEARCH & BUSINESS FOUNDATION SUNGKYUNKWAN UNIVERSITY <120> Composition for a cancer containing LOB3 <130> MP16-384 <150> KR 10-2015-0116636 <151> 2015-08-19 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer of bcl2 <400> 1 cacccctggc atcttctcct t 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of bcl2 <400> 2 cacaatcctc ccccagttca cc 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer of p53 <400> 3 ctctgtcatc ttccgtccct tc 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of p53 <400> 4 aggacaggca caaacacgaa c 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of beta-actin <400> 5 gttaccaggg ctgccttctc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of beta-actin <400> 6 gatggtgatg ggtttcccgt 20

Claims (8)

A pharmaceutical composition for treating or preventing cancer, which comprises a cyclic leucovin B3 ([1-9-N? C] -linusorb B3) as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the cyclic leucovorin B3 induces apoptosis of cancer cells.
2. The pharmaceutical composition according to claim 1, wherein the cyclic leucovin B3 reduces the activity of the cancer inducing proteins Src and STAT3.
The pharmaceutical composition according to claim 1, wherein the cyclic leucovin B3 increases the activity of Caspase-3 protein in cancer cells and induces apoptosis.
The pharmaceutical composition according to claim 1, wherein the cyclic leucovorin B3 inhibits the expression of Bcl2 and p53.
The pharmaceutical composition according to claim 1, wherein the cyclic leucovorin B3 is derived from flaxseed.
2. The pharmaceutical composition according to claim 1, wherein the cancer is a glioma.
A health functional food composition for preventing or ameliorating cancer, which comprises a cyclic leucovin B3 ([1-9-N? C] -linusorb B3) as an active ingredient.

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