KR101309538B1 - A Composition containing Eckol for inhibiting a Growth of Cancer Stem Cells - Google Patents

Abstract

The present invention relates to a cancer stem cell growth inhibitor containing Eckol compound as an active ingredient, more specifically, cancer stem by inhibiting PI3K signaling activity and Ras signal through the Ras-Raf-ERK signaling system The present invention relates to the use of Dieckol compounds to inhibit cell maintenance, malignance and invasive activity.
The exec compound is an anticancer adjuvant according to cancer stem cell inhibition, and may be usefully used for the treatment and prevention of cancer diseases.

Description

A composition containing Eckol for inhibiting a Growth of Cancer Stem Cells}

The present invention relates to the use of cancer stem cell growth inhibition containing Eckol compound as an active ingredient.

All tissues in the human body are derived from organ-specific stem cells. Organ-specific stem cells are capable of self renewal and have the ability to differentiate into all the cells that make up each organ. These organ-specific stem cells are distinguished from embryonic stem cells in that differentiation forms can mainly be differentiated only into cell types within a given organ.

The cancer stem cell hypothesis consists of two components. First, tumors arise from tissue stem cells. Second, the tumors generated and constructed from the cells must have the basic characteristics of stem cells.

Cancer stem cells are cancer cells with unlimited regenerative capacity. The hypothesis that tumors originate from stem cells has already been proposed by Cohnheim in 1875, but the cancer stem cell hypothesis has only been materialized in recent years as stem cell biology developed. It started to be. In 1997, Bonnet and Dick introduced a group of cells that could become cancer stem cells in acute myeloid leukemia and transplanted them into immunosuppressive mice, and the human stem cell hypothesis was advanced.Al-Hajj et al. By proving cancer stem cells in these breast cancers, we have confirmed the existence of stem cells in solid carcinomas.

The diverse heterogeneity of malignant tumors coincides with the differentiation of stem cells, and despite many targeted therapies, the drug resistance of cancer cells, which are constantly expressed, is consistent with the basic characteristics of stem cells. It is speculated that cancer stem cells could be a new target therapy field.

Conventional chemotherapy, chemotherapy, is one of the most common and effective treatment tools for treating cancer today, either alone or in combination with other therapies (such as radiotherapy). The efficacy of drugs used to treat cancer in chemotherapy depends on the ability to kill cancer cells. However, in the use of such drugs, there is a limitation from the fact that they can function not only in cancer cells but also in general cells that are not cancer cells.

The interest in cancer stem cells is that chemotherapy fails to target and treat these cell groups in tumors, resulting in the failure of chemotherapy and the recurrence of tumors. Many cytotoxic anticancer drugs usually target fast-growing cells, so perhaps the response to the drug was actually a response to transitional amplification cells, and cancer stem cells with slow-proliferating features could survive cytotoxic chemotherapy. will be. Basal cell phenotype Breast cancer is believed to originate from early mammary progenitor cells in the early stages of differentiation, and is known to have a poor prognosis and to be resistant to conventional chemotherapy. This is a good example to support the fact that the target treatment for cancer stem cells was not.

Based on the cancer stem cell hypothesis, several treatment methods have been devised. Among them, many known methods use the self-renewal pathway of cancer stem cells. The important point in this treatment is to target only cancer stem cell self-renewal while maintaining normal stem cell self-renewal. For example, Notch signaling is carried out by an enzyme called -secretase, which can be used to suppress tumors by using -secretase inhibitors in breast cancers that overexpress Notch1. Recently, targeting the hedgehog signaling system has been shown to be anti-cancer effect. When the hedgehog inhibitor cyclopamine was administered to a tumor xenograft animal, the tumor dramatically contracted.

However, studies on cancer stem cells have been limited so far, and their role in tumor formation and maintenance has not been clarified yet.

In order to efficiently perform treatments targeting cancer stem cells without damaging normal stem cells, knowledge and understanding of molecular biological characteristics and its regulatory pathways that are important for the maintenance and regulation of cancer stem cells are required.

To date, there is little research on anticancer drugs or natural extracts that directly target cancer stem cells. In the prior art, studies have been conducted to inhibit cancer stem cells by inhibiting cancer stem cells or inhibiting higher signaling proteins of cancer stem cells in experiments that directly inhibit target stem genes of cancer stem cells. However, in many tumor patients, this targeting experiment is difficult due to oncogene mutation or protein mutation.

Thus, improving the selectivity of drugs against cancer stem cells will definitely make it possible to use drugs at lower doses by increasing the efficacy of chemotherapy with anticancer drugs. Therefore, there is a need for an improved approach that can selectively inhibit the growth of cancer stem cells in combination with anticancer drugs for the treatment and prevention of cancer.

Therefore, the inventors of the present invention not only inhibit the expression of the brain cancer cancer stem cell markers Nestin, CD133, Musashi-1 and Sox2, Nocth2, β-catenin, which are expressions of tumor cells, By inhibiting the activity of matrix metalloproteinase (MMP) -2/9 enzymes involved in metastasis and invasion, it was confirmed that apoptosis in brain cancer stem cells increased significantly in combination with anticancer agents. The present invention was completed by elucidating molecular mechanisms related to PI3K signaling activity inhibition and Ras signal inhibition in Ras-Raf-ERK signaling system.

An object of the present invention is to provide a use of the ecol compound to inhibit the growth of cancer stem cells. That is, the present invention provides a composition for inhibiting cancer stem cell growth containing an ecol compound as an active ingredient, and a method for inhibiting cancer stem cell growth comprising treating an ecol compound.

Another object of the present invention is to provide an anticancer adjuvant containing an equol compound as an active ingredient, which enhances the ability of an anticancer drug to kill cells.

It is still another object of the present invention to provide a method for inhibiting PI3K signaling activity and Ras signal using an ecol compound.

In order to achieve the above object, the present invention provides a composition for inhibiting cancer stem cell growth containing Eckol compound of Formula 1 as an active ingredient. In another aspect, the present invention provides a method for inhibiting cancer stem cell growth, which comprises treating an equol compound of Formula 1 or a derivative thereof.

[Formula 1]

The eckol compound may be derived from brown algae, preferably Eisenia or Ecklonia. For example, Eisenia bicyclis, Eisenia arborea, Eisenia desmarestioides, Eisenia galapagensis, Icesenia Eisenia masonii, Eclonia kurome, Eclonia cava, Eclonia stolonifera, Eclonia maxima, Eclonia radiata , Eclonia bicyclis, Eclonia biruncinate, Eclonia buccinalis, Eclonia caepaestipes, Eclonia exasperta ), Eclonia fastigiata, Eclonia brevipes, Eclonia arborea, Eclonia latifolia klonia latifolia), Eclonia muratii, Eclonia radicosa, Eclonia richardiana or Eclonia wrightii.

The cancer stem cells are undifferentiated cells having the ability to differentiate into various cancer cells, and the cancer is carcinoma, lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine. Neoendocrine tumor, mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, leukemia, lymphoidmalignancy, squamous cell carcinoma ), Squamous cell carcinoma (epithelial squamous cell cancer), lung cancer (lung cancer), small-cell lung cancer, non-small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, Squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, brain cancer , Glioblastoma Cervical cancer, ovarian cancer, liver cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, Colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney and renal cancer, prostate cancer, vulval cancer, thyroid cancer (thyroid cancer), hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, biliary tract and head and neck cancer ( head and neck cancer). Preferably they are cancer stem cells with the ability to differentiate into brain cancer or glioblastoma.

The cancer stem cell growth inhibition is meant to include cancer stem cell maintenance (mainternance) inhibition, cancer stem cell malignance (inhibition) and cancer stem cell invasive activity (invasive) inhibition.

Meanwhile, the eckol compound of the present invention is characterized by inhibiting PI3K signaling activity and inhibiting Ras signal through Ras-Raf-ERK signaling system. The influence of Ackall on such a signaling system is shown in FIG.

That is, through the molecular mechanism, the exec compound of the present invention has a function of inhibiting expression of brain cancer stem cell markers Nestin, CD133, Musashi-1, and expression of Sox2, Nocth2, β-catenin, which are self-renewal markers.

In addition, by inhibiting the activity of matrix metalloproteinase (MMP) -2/9 enzymes involved in metastasis and invasion of tumor cells, it also has a function of reducing the invasive capacity of cancer stem cells.

In particular, the exec compound of the present invention is preferably treated (administered) in combination with an anticancer drug.

The anticancer drug may be selected from the group consisting of the following (a)-(x).

(a) mustard gas derivatives: mechlorethamine, cyclophosphamide, chlorambucil, melphalan, and ifosfamide;

(b) ethyleneimines: thiotepa and hexamethylmelamine;

(c) alkylsulfonates: busulfan;

(d) hydrazines and triazines: altretamine, procarbazine, dacarbazine and temozolomide;

(e) nitrosureas: carmustine, lomustine and streptozosin;

(f) metal salts: carboplatin, cisplatin and oxaliplatin;

(g) vinca alkaloids: vincristine, vinblastine and vinorelbine;

(h) taxanes: paclitaxel and docetaxel;

(i) grapephytotoxins: etoposide and tenisopide;

(j) camptothecan analogs: irinotecan and topotecan;

(k) anthracyclines: doxorubicin, daunorubicin, epirubicin, mitoxantrone and idarubicin;

(l) chromomycins: dactinomycin and plicamycin;

(m) various antitumor antibiotics: mitomycin and bleomycin;

(n) folic acid antagonists: methotrexate;

(o) pyrimidine antagonists: 5-fluorouracil, fauxuridine, cytarabine, capecitabine and gemcitabine;

(p) purine antagonists: 6-mercaptopurine and 6-thioguanine;

(q) adenosine deaminase inhibitors: cladribine, fludarabine, ellarabine and pentostatin;

(r) topoisomerase I inhibitors: ironotecan and topotecan;

(s) topoisomerase II inhibitors: amsacrine, etoposide, etoposide phosphate and teniposide;

(t) ribonucleotide reductase inhibitors: hydroxyurea;

(u) adrenocortical steroid inhibitors: mitotan;

(v) enzymes: asparaginase and pegasparase;

(w) antimicrobial agents: esthramustine;

(x) retinoids: bexarotene, isotretinoin and tretinoin (ATRA).

In one embodiment of the present invention, temozolomide was used.

Accordingly, the present invention provides an anticancer adjuvant containing an exec compound of Formula 1 or a derivative thereof as an active ingredient, which enhances the ability of an anticancer drug to kill cells.

Eckole compound derived from brown algae, by inhibiting PI3K signaling activity and Ras signaling through the Ras-Raf-ERK signaling system, selectively inhibit the growth of cancer stem cells, inhibiting malignant traits and self-renewal In combination with radiation therapy and anticancer drugs to maximize the treatment of the cancer can exhibit a therapeutic effect.

Accordingly, the exec compound is an anticancer adjuvant according to cancer stem cell inhibition and may be usefully used for the treatment and prevention of cancer diseases.

Figure 1 is a graph of FACS analysis results for apoptosis when treated by concentration (Eckol) by concentration.
Figure 2 shows the results of confirming the size (size) of cancer stem cells after the treatment.
Figure 3 shows the results of FACS analysis and Western blot analysis of the protein expression of brain cancer cancer stem cell markers Nestin, CD133, Musashi-1.
Figure 4 is an immunofluorescence stain (immunofluorescence stain) results for the protein expression of brain cancer stem cell markers Nestin, CD133, Musashi-1.
Figure 5 shows the results of immunofluorescence staining and Western blot analysis of the protein expression of Sox2, Nocth2, β-catenin as a self-renewal marker and Tuj-1 as a differentiation marker.
Figure 6 shows the results of FACS analysis for apoptosis when treated with radiation (6Gy) and anticancer drug (temozolomide).
Figure 7 is a soft agar assay results (A) to determine the malignant traits of tumors in the in-vitro and observation results of the tumor formation ability of the exol for nude mice.
8 is a result of Western blot analysis of the results of invasion assay and MMP (matrix metalloproteinase) -2/9 enzyme activity involved in confirming the invasive activity of cancer stem cells.
FIG. 9 is a Western blot result for kinase experiment and Raf-1 activity experiment to investigate the effects of Eckol on PI3Kinase signaling activity and Ras-Raf-ERK signaling mechanism activity.
10 is a schematic diagram of the molecular mechanisms involved in PI3Kinase signaling activity and Ras-Raf-ERK signaling activity of Eckol.
11 and 12 are FACS results, Western blotting results and immunofluorescence staining results confirming the formation of cancer stem cells from brain tumor cells.

The terms used in the present invention are defined as follows.

"Stem cell " means a cell capable of developing into any tissue. There are two basic features: self-renewal, which creates itself by repeated division, and the ability to differentiate into cells with specific functions depending on the environment.

The term "differentiation" refers to a phenomenon in which cells and tissues specialize in structure or function, while cells grow and proliferate, that is, the form or function changes to carry out the task given to each of them . In general, a relatively simple system is separated into two or more qualitatively different systems. For example, there may be qualitative or quantitative differences between parts of a biological system that were initially homogeneous, such as head or trunk distinction between eggs that were initially homogeneous in origin, or distinctions of cells such as muscle cells or neurons The state in which there is a difference or as a result is divided into qualitative or inferior parts or partial systems is called differentiation.

"Undifferentiated" means a property that represents an undifferentiated state that can be confirmed by expression of at least one or more undifferentiated markers or various antigen molecules in stem cells. An undifferentiated state of stem cells means a state in which stem cells are capable of proliferating almost permanently or for a long time, exhibit a normal nucleus (chromosome) type, and have the ability to differentiate into cells of all lineages of the three germ layers under appropriate conditions.

The term "proliferation" or "growth" of a cell refers to a cell that divides and is homogeneous, usually increasing in the body of a multicellular organism. In time, it is common for traits to be changed (differentiated) and controlled at the same time.

"Cell death" is used broadly and refers to a mammal that is generally accompanied by one or more characteristic cell changes, including loss of cytoplasmic compression, loss of plasma membrane microvilli, breakdown of nuclei, degradation of chromosomal DNA, or loss of mitochondrial function It means a controlled or controlled cell lethality.

"Cancer "," tumor ", or "malignant" refers to or represents the physiological condition of a mammal that is generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma.

An "inhibitor" or "inhibitor" refers to a substance that inhibits, blocks or reduces the activity associated with the growth of cancer stem cells. The mechanism of activation of the inhibitor is not particularly limited. For example, 'cancer stem cell inhibitor' may include a substance that inhibits, blocks or reduces the activity of cancer stem cells in tumorigenicity.

"Treatment" means an approach to obtain beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected. "Treatment" may also mean increasing survival compared to expected survival when untreated. "Treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Such treatments include the treatments required for the disorders that have already occurred as well as the disorders to be prevented. "Palliating" a disease may reduce the extent of the disease state and / or undesirable clinical signs and / or slow or lengthen the time course of progression as compared to untreated treatment. It means losing.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention.

Hereinafter, the present invention will be described in detail.

Eckol, a type of polyphenolic compound, phlorotannins, is known to inhibit intracellular free radicals and to inhibit apoptosis of normal cells during radiation therapy. Known. In addition, there have been reports of inhibiting collagen destruction by inhibiting matrix metalloproteinase-1 (MMP-1) activity in skin fibroblasts, thereby inhibiting phenotype of skin cancer. There is no.

The present invention relates to a novel use of the Eckol compound, the inventors first identified that it has an anticancer function against cancer stem cells.

In particular, dieckol, which inhibits PI3K signaling activity and inhibits Ras signaling through the Ras-Raf-ERK signaling system, inhibits maintenance, malignance and invasive activity of cancer stem cells. The present invention relates to a selective anticancer use of the compound against cancer stem cells.

“Optionally” refers to the ability of a compound of the invention to inhibit the growth of cancer stem cells without affecting non-cancer stem cells at a given concentration.

The present invention relates to a cancer stem cell growth inhibitory use of the excol compound represented by the following formula (1) and derivatives thereof in one aspect. That is, the present invention relates to a method for inhibiting cancer stem cell growth comprising an Eckol compound of Formula 1 as an active ingredient and / or a method for inhibiting cancer stem cell growth comprising treating an Eckol compound of Formula 1 or a derivative thereof. will be.

The ecol compound of the present invention may be prepared by separating the compound from brown algae, in particular from the Eisenia or Ecklonia family.

Specifically for the brown algae, Eisenia bicyclis, Eisenia arborea, Eisenia desmarestioides, Esenia galapagensis, Eisenia masonii, Eclonia kurome, Eclonia cava, Eclonia stolonifera, Eclonia maxima, Eclonia radiata Ecklonia radiata, Eclonia bicyclis, Eclonia biruncinate, Eclonia buccinalis, Eclonia caepaestipes, Eclonia exasperta (Ecklonia exasperta), Eclonia fastigiata, Eclonia brevipes, Eclonia arborea, Eclonia latifolia, Eclonia muratii, Eclonia radicosa, Eclonia richardiana or Eclonia wrightii can be used, most Preferably Ecklonia cava can be used.

The ecol compound may be prepared and used by a method known in the art, a modified method thereof, or a method according to the present invention, and may be purchased and used commercially.

Eccol compound according to the present invention

(a) extracting the eclonia carba powder with an organic solvent; And

(B) by fractionating the organic solvent extract can be prepared by a method comprising the step of separating and purifying the compound by chromatography.

As the organic solvent in step (a), ethanol, methanol and the like can be used, preferably methanol.

In one embodiment of the present invention E. cava can be used without limitation, such as cultivated or commercially available, it is used to clean and dry. The dried powder of E. cava is extracted with an appropriate amount of an organic solvent, preferably methanol, and the obtained methanol extract is fractionated with ethyl acetate. The exol compound according to the present invention is isolated and purified by chromatography using a stepwise gradient of the solvent.

The preparation method is merely an exemplary method thereof, and may be suitably modified by various methods based on the art. For example, the separation and purification of non-exemplified compounds according to the present invention Modifications apparent to those skilled in the art can be successfully carried out, for example, by appropriately protecting the interferer, replacing it with another suitable reagent known in the art, or by customarily changing the reaction conditions.

The extract compound of Formula 1 of the present invention obtainable by the above method has a selective growth inhibitory function on cancer stem cells. The 'growth inhibition' includes the maintenance of cancer stem cells and the inhibition of self-renwal.

Recently, cancer stem cells, which regenerate and maintain cancer tissues, also exist in malignant tumor tissues similar to normal stem cells, and these cancer stem cells are resistant to cancer treatment and are not extinguished without specific signals. The cancer stem cell hypothesis has been attracting attention as it contributes to the regeneration or metastasis of cancer by integrating cancer cells that have been reduced after treatment.

Cancer stem cells are stem cells that have the ability to differentiate into various cancer cells with unlimited regenerative capacity and should have the basic characteristics of stem cells.

The term "cancer" refers to or refers to a physiological condition characterized by cell growth that is not typically regulated in mammals. Cancers to be treated and prevented include carcinoma, lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma, and schwanoma. ), Meningioma, adenocarcinoma, melanoma, leukemia, lymphoidmalignancy, squamous cell cancer, epithelial squamous cell cancer, lung cancer (lung cancer), small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, peritoneum ( cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, brain cancer, glioblastoma, cervical cancer, Ovarian cancer, liver cancer r), bladder cancer, hepatoma, breast cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, Salivary gland carcinoma, kidney and renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatocarcinoma, anal carcinoma , Penile carcinoma, testicular cancer, esophageal cancer, biliary tract and head and neck cancer. Preferably, the cancer is brain cancer or glioblastoma.

Cancer stem cells capable of differentiating into these cancer cells are present in the malignant tumor tissue in about 1-2%, and have self-renewal ability, which is characteristic of normal stem cells, and pluripotent which can differentiate into other cells. However, it has been reported to have an abnormality in self-regulatory function, thereby increasing cell number and differentiating itself into malignant tumor cells.

Due to the characteristics of the cancer stem cells, the general cancer cells are removed through chemotherapy, but the cancer stem cells survive, and it has been found that some cancer stem cells survive and the cancer recurs and metastasizes.

Since the presence of cancer stem cells in leukemia by Dr. John E. Dick in 1997 (Blood, 1997), breast cancer (PNAS, 2003), brain tumors (Nature, 2004), prostate cancer (Cancer Res, 2005 Evidence of cancer stem cells is also present in colorectal cancer (Nature, 2007) and melanoma (Nature, 2008). A small number of cancer stem cells in tumors have emerged as the main cause of tumor malignancy, anticancer resistance and recurrence.

To distinguish cancer stem cells from other cancer cells, only markers of cancer stem cells are required. Cancer stem cell markers (cancer stem cell markers) are currently known cancer-specific cancer stem cell markers in a variety of human carcinoma as follows.

Typically, the following cancer stem cell markers may be used as markers.

- glioblastoma: CD133

Acute myeloid leukemia (AMM): CD34 + / CD38- (9)

Multiple myeloma: CD133- (10)

Breast cancer: CD44 + / CD24- / low (11)

- Colon cancer: CD133 + (12,13)

- prostate cancer: CD44 + /? 2? 1hi / CD133 +

Melanoma: ABCB5 + (14)

In particular, one embodiment of the present invention targets the brain cancer stem cells, including glioblastoma.

Cancer stem cells continually self-renewal and can produce tumors with fewer than a thousand cells in experimental animal models and possess the capacity of malignant tumor cells. In addition, it is surprisingly resistant to anticancer and radiation therapy, which is a cancer treatment method, and the removal of cancer stem cells is increasingly recognized as a barometer for measuring the success or failure of cancer treatment. Recently, even if cancer cells are killed using various treatment methods such as surgery, radiation therapy and chemotherapy, it is recognized that cancer may recur from remaining cancer stem cells if the cancer stem cells are not killed. have. In order to prevent the recurrence of these cancers, there is increasing interest in the development of chemotherapy targeting cancer stem cells having the ability to regenerate tumors and the treatment protocol to treat cancer based on the same.

Stem cells in normal tissues regulate cell growth and differentiation by self renewal mechanisms, but cancer stem cells are affected by tumor microenvironment factors surrounding tumor cells, resulting in abnormal self-renewal and maintenance. It has been suggested to activate the maintenance pathway and accumulate rapidly, thereby becoming malignant, obtaining resistance to chemotherapy, and ultimately causing cancer to recur. However, there are no specific mechanisms yet to study the reality and interaction of tumor microenvironment factors that regulate the accumulation and maintenance of cancer stem cells.

Accordingly, the exec compound of the present invention enables chemotherapy to selectively target cancer stem cells, thereby providing a more effective anticancer treatment method that can prevent cancer from recurring.

In particular, by using in combination with existing cancer treatment method can maximize the cancer treatment effect.

Anticancer drugs or radiation therapy used to treat cancer are known to damage normal cells and have various side effects. However, Eckol does not affect normal cells at all and shows selective growth inhibition only to cancer stem cells. Therefore, by using a combination of conventional anticancer or radiation therapy, Eckol induces cancer cell death and cancer stem cell death by inducing a combination of cancer cells. The anticancer resistance and radiation resistance, which is a problem of cancer treatment, can be reduced to play a significant role in cancer treatment.

Eckole compound of the present invention inhibits the growth of cancer stem cells by the following molecular mechanisms. This molecular mechanism is shown schematically in FIG. 10.

(1) The exec compound of Chemical Formula 1 of the present invention inhibits PI3K signaling activity.

PI3K (phosphatidylinositol-3-kinase) plays a major role in cell proliferation and survival and glucose metabolism in phospholipid metabolism of cell membranes, and is associated with the overexpression of p85 in malignant tumor cells.

When PI3K is activated, it performs functions related to cell growth and survival, glucose metabolism and protein interpretation through serine-threonine protein kinase Akt (or protein kinase B, PKB). Lipid products produced by PI3 kinase bind to some proteins and affect their intracellular location and activity.

Various studies have shown that pleckstrinhomology (PH) and C2 domains can bind to lipids, and some Ser / Thr kinases have such domains, which are placed in the lower stage of PI3kinase. Akt / PKB, PtdIns, and P2 / P3-dependent kinase 1 (PDK1). Akt / PKB is an intracellular analogue of v-akt that has a PH-domain and is expected to be involved in phosphorylation of various downstream targets by receiving signals from PI3 kinase.

Mitogen-activated protein kinase (MAPK) plays a decisive role in regulating cell growth and differentiation, and is affected by cellular responses to cytokines and stress. Inhibition of PI3 kinase activity by exol according to the present invention It may also be mediated via the MAPK path.

Therefore, Eckol according to the present invention inhibits the activity of MAPK through the mechanism of inhibiting the activity of PI3 kinase involved in phosphorylation of various lower targets affects the growth and differentiation of cancer stem cells. That is, it suppresses maintenance and malignance of cancer stem cells.

Through this, expression of stem cell markers and self-renewing marker genes associated with maintenance of cancer stem cells is suppressed. In one embodiment of the present invention, the inhibition of expression of the brain cancer stem cell markers Nestin, CD133, Musashi-1 and the inhibition of expression of Sox2, Nocth2, β-catenin, which are self-renewal markers, were confirmed.

The ecol compound according to the present invention exhibits the same effect as LY294002, an inhibitor of PI3K, which is known in the art (Example 5).

(2) The extract compound of Formula 1 of the present invention inhibits the Ras signal through the Ras-Raf-ERK signaling system.

Together with PI3K / AKT, Ras / Raf / MAPK signaling pathways affect cell division and survival in the nucleus, angiogenesis and ultimately metastasis and invasion into surrounding tissues. For this reason, understanding and blocking Ras signaling pathways may be more important in the treatment of cancer.

Ras is a representative cancer protein that inhibits cell proliferation and apoptosis and is GTPases bound to the cell membrane. To date, no drugs that can directly block ras have been developed, and drugs that block the lower signaling system by Ras by blocking farnesylation, which is critical for ras activation, have been developed.

Activation of the Raf protein is accompanied by phosphorylation at its tyrosine, serine and threonine residues. Direct phosphorylation by receptor tyrosine kinase or phosphorylation by protein kinase regulated by these receptors is known as the mechanism of Raf activation. When regulated by receptors, Ras is involved in the activation of Raf. The signal reaching Raf is then delivered to the nucleus via a signaling pathway leading to Raf / MEK / MAPK. In this signal transduction pathway, a series of kinases are arranged in a species to transmit signals, which play an essential role in cell growth and differentiation.

The primary function of Raf protein signaling pathways is to phosphorylate and activate MEK (MAPK kinase (MAPKK)). MEK promotes phosphorylation of ERK1 and ERK2 (MAPKs), and then phosphorylated phospho-ERK (pERK) forms a dimer and transfers into the nucleus to activate transcription factors, dividing and differentiating cells, survival, invasion, and metastasis. Cause. Activated MAPK is observed at high frequency in cancer, which activates MEK continuously. This ERK / MAPK step is a good target for the treatment of many malignant tumors in that it is the step of activating transcription factors and its location itself is the last step in various activities related to cell division and cancer development.

Eckol compound of the present invention in the Ras-Raf-ERK signaling system, by inhibiting the Ras signal to suppress downstream signals, inhibits the maintenance and malignant, invasive activity of cancer stem cells.

On the other hand, the extract compound of Formula 1 of the present invention is preferably used in combination with other anticancer therapy. In other words, by using in combination with conventional radiation therapy or chemotherapy can maximize the cancer treatment effect.

In particular, by administering with a chemical anti-cancer drug and the like, through the selective targeting effect of cancer stem cells to reduce the conventional anti-cancer resistance and radiation resistance to prevent the recurrence of cancer as well as to treat the cancer at the source.

In this regard, the present invention relates to an anticancer adjuvant containing an exec compound of Formula 1 or a derivative thereof as an active ingredient, which enhances the ability of an anticancer drug to kill cells.

This ability can be measured in vitro, for example, by treating the compound of the invention with an anticancer agent and recording the number of cells showing enriched nuclei (markers of apoptotic cells) to determine the percentage of apoptosis of the cells. Typically, the results are compared to the results of treatment with only anticancer agents without treatment with the ecol compound. Compounds of the invention that result in doubling the apoptosis of cancer stem cells at a given concentration, or at least twice the dose of a drug that induces a given apoptosis response, have the ability of the drug to kill cancer stem cells. It can be judged to increase.

The anticancer drug is any drug used in the past, and there is no limitation, for example, it may be selected from the group consisting of the following (a)-(x):

(a) mustard gas derivatives: mechlorethamine, cyclophosphamide, chlorambucil, melphalan, and ifosfamide;

(b) ethyleneimines: thiotepa and hexamethylmelamine;

(c) alkylsulfonates: busulfan;

(d) hydrazines and triazines: altretamine, procarbazine, dacarbazine and temozolomide;

(e) nitrosureas: carmustine, lomustine and streptozosin;

(f) metal salts: carboplatin, cisplatin and oxaliplatin;

(g) vinca alkaloids: vincristine, vinblastine and vinorelbine;

(h) taxanes: paclitaxel and docetaxel;

(i) grapephytotoxins: etoposide and tenisopide;

(j) camptothecan analogs: irinotecan and topotecan;

(k) anthracyclines: doxorubicin, daunorubicin, epirubicin, mitoxantrone and idarubicin;

(l) chromomycins: dactinomycin and plicamycin;

(m) various antitumor antibiotics: mitomycin and bleomycin;

(n) folic acid antagonists: methotrexate;

(o) pyrimidine antagonists: 5-fluorouracil, fauxuridine, cytarabine, capecitabine and gemcitabine;

(p) purine antagonists: 6-mercaptopurine and 6-thioguanine;

(q) adenosine deaminase inhibitors: cladribine, fludarabine, ellarabine and pentostatin;

(r) topoisomerase I inhibitors: ironotecan and topotecan;

(s) topoisomerase II inhibitors: amsacrine, etoposide, etoposide phosphate and teniposide;

(t) ribonucleotide reductase inhibitors: hydroxyurea;

(u) adrenocortical steroid inhibitors: mitotan;

(v) enzymes: asparaginase and pegasparase;

(w) antimicrobial agents: esthramustine;

(x) retinoids: bexarotene, isotretinoin and tretinoin (ATRA).

In one embodiment of the present invention, temozolomide was used. "Temozolomide" means temozolomide and all analogs, pharmaceutically acceptable salts and prodrugs thereof.

Unless stated otherwise, the term "exol compound" or "compound of formula 1" according to the present invention refers to the compound itself, a pharmaceutically acceptable salt, hydrate, solvate, isomer and prodrug thereof. It is used as a concept to include all.

"Pharmaceutically acceptable salt" means a formulation of a compound that does not cause severe irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The terms "hydrate "," solvate ", and "isomer" The pharmaceutical salt may be prepared by dissolving the compound of the present invention in an organic solvent such as mineral acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid, sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, or p-toluenesulfonic acid, tartaric acid, formic acid, Can be obtained by reacting an organic carboxylic acid such as benzoic acid, benzoic acid, lactic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid and the like. In addition, the compound of the present invention is reacted with a base, and salts such as alkali metal salts such as ammonium salts, sodium or potassium salts, and alkaline earth metal salts such as calcium or magnesium salts, dicyclohexylamine, and N-methyl-D-glu It may be obtained by forming salts of organic bases such as carmine and tris (hydroxymethyl) methylamine, and amino acid salts such as arginine and lysine.

A "hydrate" is a compound of the present invention comprising a stoichiometric or non-stoichiometric amount of water bound by a non-covalent intermolecular force or It means salts.

"Solvate" means a compound of the present invention or a salt thereof comprising a stoichiometric or nonstoichiometric amount of solvent bound by non-covalent intermolecular forces. Preferred solvents therein are volatile, nontoxic, and / or solvents suitable for administration to humans.

"Isomer" means a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula, but which is optically or sterically different. For example, the compound according to Formula 1 of the present invention may have an asymmetric center (asymmetric carbon atom) depending on the kind of substituents. In this case, the compound of Formula 1 may be an enantiomer or diastereomer May exist as the same optical isomer.

"Prodrug" means a substance that is transformed into a parent drug in vivo. Prodrugs are often used because, in some cases, they are easier to administer than the parent drug. For example, they may be bioavailable by oral administration, while the parent drug may not. Prodrugs may also have improved solubility in pharmaceutical compositions over the parent drug. For example, prodrugs are esters that facilitate the passage of cell membranes, which are hydrolyzed to carboxylic acids, which are active by metabolism, once the water solubility is detrimental to mobility, but once the water solubility is beneficial. Drug "). Another example of a prodrug may be a short peptide (polyamino acid) that is attached to an acid group that is converted by metabolism so that the peptide reveals its active site.

The present invention relates to a method for treating and preventing cancer through growth inhibition of cancer stem cells using the compound 1 or a derivative compound thereof in one embodiment. That is, the exec compound of the present invention can inhibit differentiation into various cancer cells through cancer stem cell growth inhibition, and thus has a therapeutic and preventive effect on cancer.

In still another aspect, the present invention relates to a pharmaceutical composition for preventing and treating cancer by inhibiting cancer stem cells, comprising an exec compound represented by Chemical Formula 1 as an active ingredient.

The pharmaceutical composition for preventing and treating the cancer or the composition for inhibiting cancer stem cells may further include a diluent, an excipient, and the like, as necessary.

"Pharmaceutical composition" means a mixture of a compound of the invention with other chemical components such as diluents or carriers.

The pharmaceutical composition facilitates administration of the compound into the organism. Various techniques exist for administering the compounds, including, but not limited to, oral, injectable, aerosol, parenteral, and topical administration. The pharmaceutical composition may be obtained by reacting acid compounds such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

A “therapeutically effective amount” means the amount of the compound administered to alleviate to some extent one or more of the symptoms of the disorder being treated. Thus, a pharmacologically effective amount means (1) to reverse the rate of progression of a disease or (2) to prohibit further progression of the disease, and (3) to some extent one or more symptoms associated with the disease. It means the quantity which has an effect of reducing (preferably removing).

A "carrier" is defined as a compound that facilitates the addition of a compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the incorporation of many organic compounds into cells or tissues of an organism.

A "diluent" is defined as a compound that not only stabilizes the biologically active form of a compound of interest, but also is diluted in water to dissolve the compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, since it mimics the salt state of the human solution. Since buffer salts can control the pH of the solution at low concentrations, buffer diluents rarely modify the biological activity of the compounds.

"Physiologically acceptable" is defined as a carrier or diluent that does not impair the biological activity and properties of the compound.

The compounds used herein may be administered to human patients as such or as pharmaceutical compositions in combination with other active ingredients or with a suitable carrier or excipient, such as in a combination therapy.

(a) route of administration

Suitable routes of administration include parenteral delivery, including, for example, intramuscular, subcutaneous, intravenous, bone marrow injections, as well as intraoral, intranasal, transmucosal, or enteral septum, direct intraventricular, intraperitoneal, or intraocular injections. Include. Preferably parenteral delivery.

In addition, the compounds may also be administered in a local rather than systemic manner, for example by direct injection into solid tumors, often in immersion or sustained release formulations.

(b) Compositions / Formulations

Pharmaceutical compositions of the invention can be prepared in a known manner, for example, by means of conventional mixing, dissolving, granulating, sugar-making, powdering, emulsifying, encapsulating, trapping and or lyophilizing processes. .

Thus, a pharmaceutical composition for use according to the invention comprises one or more pharmacologically acceptable which comprises excipients or auxiliaries which facilitate the treatment of the active compounds into formulations which can be used pharmaceutically. It may also be prepared by conventional methods using a carrier. Suitable formulations depend on the route of administration chosen. Any of the known techniques, carriers and excipients may suitably be used.

The compounds of the present invention may be formulated for parenteral infusion by injection, for example by large pill injection or continuous infusion. Injectable formulations may be presented in unit dose form, for example, as ampoules or multi-dos containers, with preservatives added. The compositions may take such forms as suspensions, solutions, emulsions on oily or liquid vehicles, and may also contain components for the formulation, such as suspending, stabilizing and / or dispersing agents.

In addition to the formulations described above, the compounds may be formulated as deposits. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular infusion. Thus, the compounds may, for example, be formulated with a suitable polymer or hydrophobic material (such as an emulsion in an acceptable oil), or with an ion exchange resin, or as a low soluble derivative, for example a low soluble salt. have.

The formulation carrier for the hydrophobic compound of the present invention is a cosolvent system composed of benzyl alcohol, nonpolar surfactant, water-miscible organic polymer, and liquid phase. The cosolvent system may be a V palladium cosolvent system. This cosolvent system dissolves hydrophobic compounds well and provides itself with low toxicity upon systemic administration. Naturally, the proportion of cosolvent system may vary considerably without compromising its solubility and toxicological properties. Moreover, the identification of cosolvent components can be varied.

(c) effective amount

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in an amount effective to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prolong the survival of the subject to be treated or to prevent, alleviate or alleviate the symptoms of a disease. Determination of a therapeutically effective amount is within the capabilities of those skilled in the art, in particular in terms of the detailed disclosure provided herein. For systemic administration, a therapeutically effective amount or dose may be assessed initially from in vitro assays. For example, the dosage may be formulated in an animal model to reach a circulating concentration range that includes IC50 as determined in cell culture. This information can be used to more accurately determine the effective dosage for humans.

In addition, the initial dose may be assessed from in vivo data, such as animal experiments, for example using techniques well known in the art. One of ordinary skill in the art can easily optimize administration to humans based on animal data.

The exact estimate, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. Typically, the dose range of the composition administered to the patient may be about 0.5 to 1000 mg / kg of the patient's body weight. Dosages may be given in a series of two or more, one at a time or in a day or more, depending on the extent required by the patient.

Of course, the amount of composition to be administered will depend on the subject to be treated, on the weight of the subject, on the severity of the pain, on the manner of administration and on the judgment of the physician.

As described above, administration of the composition containing the iccol compound of the present invention is preferably used in combination with other anticancer therapy.

Anticancer drugs or radiation therapy used to treat cancer are known to damage normal cells and have various side effects. However, Eckol does not affect normal cells at all and shows selective growth inhibition only to cancer stem cells.

In this case, when one or more anti-cancer drugs are used, it is possible to determine a useful combination of drugs within the capabilities of those skilled in the art, depending on cancer stem cells to be killed and the like.

As such, the present invention reduces the anticancer resistance and radiation resistance, which is a problem of conventional cancer treatment, by inducing the death of cancer stem cells together with the death of cancer cells by using them in combination with existing anticancer agents or radiation treatments. Will be able to.

(Example)

The invention is illustrated in more detail by the following examples, preparations and experimental examples. However, the scope of the present invention is not limited to the following examples and the like.

Manufacturing example  1: Cancer Stem Cell Formation

After forming cancer stem cells from brain tumor cells (U87MG, U373MG Korea Cell Line Bank), mix (DMEM / F12 (-) + bFGF (20ng / ml) + hEGF (20ng / ml) + B27 stem cell suppliment (1X)) Incubated in one media.

The formed cancer stem cells, Papulation of CD133 was analyzed using FACS, and the expression patterns of cancer stem cell markers CD133, Nestin, Musashi-1 were confirmed by Western Bloting experiment. This is illustrated in FIGS. 11 and 12. About U373MG, it was confirmed by immunofluorescence staining (immunofluorescence stain) and shown in FIG. 12.

In all examples, the cells were treated with EXCOL (obtained from Jeju National University) for a defined time period.

Experimental Example  1: various experimental methods

(One) FACS ( Fluorescence Activated Cell Sorting ) analysis

1) MACS

As a method for separating magnetically activated cells, positive (+) and negative (-) of CD133, stem-like cell markers, were separated.

The sample was washed with 2 ml of PBS, incubated for 5 minutes after adding 10 ml of 10 × T. 1 ml of the solution was taken and dished up & down, and then put back into the tube and centrifuged at 2000 rpm for 3 minutes. After removing the supernatant and injecting 70 μl of blocking reagent into the ice bath for 5 minutes, 30 μl of CD133 microbeads were added (fluidized) and stored in the ice bath for 30 minutes. Centrifuged for 10 minutes per 300g and put 1ml of PBS in the tube and then up & down.

After preparing the MACS multistand magnetic filter and two E-tubes, 1 ml of PBS was added to the pellet, and the magnetic filter was pressed down to receive the tubes. After that, put 1ml of PBS into the syringe and just press to get a positive. After centrifuge for 3 minutes at 2000rpm, the supernatant was suctioned, washed with 1ml of 1% BSA, centrifuge again at 2000rpm for 3 minutes, and the supernatant was suctioned.

15 μl of CD133 / (293c3) -PE (staining) for FACS was injected and stored in an ice bath for 30 minutes. 400 μl of PBS was added to the tube after up & down, and CD133 + population was confirmed by FACS analysis.

2) Cell Death

A sample called PI was used to see cell death. PI is a substance that stains the nucleus, and because the cell does not pass through the cell membrane of the living cell, the living cell is negative, and the dead cell opens the cell membrane.

After treatment with the drug, 72 hours after the sample was removed from the incubator, each was transferred to a 15ml tube, and the dish was washed with 1ml of 1 × PBS and collected in a 15ml tube. 500 μl of Trypsin-EDTA was injected into each dish and stored in the incubator for 5 minutes. After trypsin-treated dish up and down with a solution in 15ml, put it in a 15ml tube and spin down at 2000rpm for 3 minutes, leaving only pellet. Media were suctioned and sampled by 500 μl each with PI. PI was diluted in 1 × PBS and 1000: 1 ratio (1 μl per ml). (500 μl PBS per sample was used.) The stained cells were transferred to FACS tubes, labeled, and placed in a test tube. Put on foil and put FACS on it.

(2) Western Blot  analysis

The cells in the dish treated with the drug-treated cancer stem cells were scraped with a scraper and collected in a 15 ml tube with media. 1 × PBS 1-2ml was placed in a dish and scraped again, collected together in a tube, followed by spin down at 2000 rpm for 3 minutes. Suction the media in the upper part, put 1ml of 1 × PBS into the tube, loosen the cell, transfer it to the 1.5ml tube, shake well, spin down at 2000rpm for 3 minutes, and suction the supernatant.

-Sampling

1 ml of inhibitor (Aprotinin, Leupeptin, AEBSF, Na3VO4) was mixed in 1 ml of TNN (-) solution to make a cell lysis buffer, and then vortexed in 15 ml tubes and injected in an appropriate amount according to the amount of cells. The tube containing the TNN (+) solution was kept in an ice bath for 30 minutes and vortexed at 5 minute intervals, followed by spin down at 13200 rpm (4 ° C.) for 20 minutes to collect the supernatant in a micro tube.

Bio-rad protein assay (Bradford reagent)

 Protein measure solution: After mixing and diluting at a ratio of DW = 1: 4, 5 ml of standard and 1 ml of each tube were added to each tube. BSA (1mg / ml) was added to 5 standard tubes, 0, 3, 6, 9, and 12µl, respectively, and 2µl of protein was added to the rest, followed by vortexing. 150 μl of the solution prepared in 96 wells was measured, and then the concentration was measured using an AbS measuring machine, and the amount of protein used was calculated. Protein, TNN solution (made cell lysis buffer) and 4 × SSB (s-s bond breaker) were added according to the calculated amount, and then heated at 94 ° C. for 5 minutes after vortexing.

-gel plate making

The glass plate was washed with ethanol and fixed, depending on the percentage of H2O, Acryl / bis-Acryl, and 3MTris-HCl.

A resolution gel made by mixing 10% SDS, 10% APS and TEMED was poured, leaving about 2cm above the gel plate. If you put Temed, it starts to harden, so I put it at the end. In order to remove the foam from the gel, DW 500ml was added directly on top of it. After removing the DW, the stacking gel was injected and the comb was plugged in. The comb acts as a well when loading proteins.

-Electrophoresis

 The process of separating proteins by size using gel. Denature the protein and then process it with SDS. SDS surrounds the protein and creates negative charges proportional to the surface. The difference in speed is the principle of electrophoresis. Therefore, electricity is charged from top to bottom.

Running buffer was prepared by mixing 50 ml of 10 × DGB and 2.5 ml of 20% SDS in 450 ml of D.W. After removing the comb of the gel prepared in advance, wash the formed well with DW, install it on the phor device and pour the running buffer. Samples were prepared with markers by spin down after vortexing. At the far left of the well, a marker (a measure of molecular weight of proteins and an indicator of the degree of migration) was put in about 30 μl and samples were loaded in order. The final well was loaded with 1 × sample and electrophoresed for 120 min at 120 V and 400 mA. When the sample reached the tip, the power was turned off and the glass plate was removed.

-transfer

 Electrophoresis is the process of transferring the separated protein to the membrane to see it well. The principle is the same as electrophoresis, and electricity is applied from front to back ((-) pole → (+) pole).

 A transfer buffer was prepared by mixing 200 ml of methanol and 100 ml of 10 × DGB in DW 700 ml. After the gel was removed from the glass plate and cut 5mm below. Wet gel, membrane, and 3M paper separated with transfer buffer and set in the kit. The order of the transparent plate, sponge, 3M paper, gel, membrane, 3M paper, sponge, the black plate of the kit in the order. Place the gel so that the maker is on the left side, set the ice so that it is in front of the black plate of the kit, pour the transfer buffer and transfer for 60 minutes at 90V, 400mA. At the end of the transfer, the device was released, the membrane was separated, placed in a container, and stained with ponceau S solution for 5 minutes. When the protein stained red, the solution was decanted and washed 2-3 times with 5% acetic acid. After removing the water, check the size and write the name of the desired protein and cut it. Since the makers are labeled with molecular weight, they can be distinguished by their molecular weight.

antibody attaching

 Proteins were labeled using antibodies to help identify them. The cut membrane was washed with shaker 1 × PBST on the shaker, and when the color was gone, 5% skim milk (25g skim milk powder, 50ml 1 × PBST) was poured and shaken for 10-20 minutes. The primary antibody prepared by adding 5 µl of primary antibody (1: 1000) to 5 ml of 5% skim milk was placed in a container with membrane for each protein and shaken overnight at 4 ° C chamber.

The membrane cut according to whether the secondary antibody to be used is rabbit, mouse and goat was separated and washed with 1 × PBST for 15 minutes at 5 minutes intervals and 30 minutes at 10 minutes intervals for rabbits. Add 1 μl of the secondary antibody to 10 ml of 5% skim milk, pour the diluted antibody into the separated membrane, shake it for 1 to 2 hours, pour out the antibody, and wash for 30 minutes at 10 minute intervals using 1 × PBST. Did

-Develope

Western lighting reagents such as luminol reagent and oxidizing reagent were vortexed in the amount of 1: 1 in the tube, and the membrane was put on the film and evenly buried. After the membrane solution was removed using a tissue, the membrane was transferred to a cassette, covered with OHP paper, and pressed. At this point, you should rub well to let air out of the film. The film was developed in the dark.

(3) immunofluorescence analysis

1cm × 1cm cover glass was put on the 35mm dish and the cell was put. After 72 hours of treatment with Eckol, the media was suctioned. In the case of the monolayer, 800 μl of 4% pormaldehyde was added to fix the cell and placed at room temperature for 1 hour. Then, washed with PBS × 3, 1ml of blocking solution was put at room temperature for 30 minutes. After washing twice with PBST for 10 minutes, the tip was removed and the primary antibodies (CD133, Nestin, Musashi-1, Tuj1, Sox2) were detected. A second antibody (FITC-mouse, rabbit) was prepared by mixing 5 μl of antibody per 1 ml of blocking solution. 300 ㎕ of each well was put on a silver foil and shaken for 2 hours. The tip was suctioned with liquid and washed with PBS for 10 minutes. 1 ml of PBS was mixed per 1 μl of Dapi solution, and 1 ml of each solution was added to a 35 mm dish, waited for 5 minutes, washed with PBS for 10 minutes, and observed with a fluorescence microscope.

In the case of glioma stem-like cells, a solution mixed with MeOH: acetic acid = 3: 1 was added and fixed for 10 minutes. After adding the blocking solution and stored at room temperature for 30 minutes was carried out in the same manner as Monolayer.

(4) soft agar analysis soft agar assay )

Mixing 1.8% soft agar and 2 × DMEM / F12 (-) in a 1: 1 ratio hardens like a jelly, so 3 ml of a 60mm dish was hardened on a bench to make agar. Cells were counted (5X104 cells / ml) and the amount considering the number of dishes was put in a 1: 1 mixture of 2 × DMEM / F12 (-) and 0.9% agar and cooled at 37 ° C. 0.9% agar and 3ml each of the sample on the support agar, put the new DMEM / F12 (+) when the agar plate cooled, and then incubated for 12-30 days in a 5% CO2 incubator at 37 ℃. Colonies with a diameter of at least 0.1 mm were counted under the microscope at x40 magnification.

(5) PI3Kinase assay

 After collecting the cells, perform Bio-rad protein measure (300ug / 300µl), then 1% (v / v) NP-40, 10% (v / v) glycerol, 137 mmol / L NaCl, 20 mmol / L Tris- MIX with HCl (pH 7.4), 1% (v / v) Triton X-100, and protease inhibitors. 1 μl of P85 antibody was added and 4 ° C. overnight rotary shaking was performed. 100 μl of protein beads were added to the cell and reacted at 4 ° C. for 2 hours, followed by washing with 100 mM Tris-HCl (ph 7.5) + 5 mM LiCl + 0.1 mM Na3VO4 + autoDW solution to carry out Centrifuge (3000 rpm / 3 min). Thereafter, the mixture was washed with 50 mM Tris-HCl (ph 7.2) + 150 mM NaCl + 0.1 mM Na3VO4 + autoDW solution and centrifuged (3000 rpm / 3 min).

The supernatant was completely removed by washing with 20 mM HEPES (ph7.2) + 100 mM NaCl + 0.1 M Na3VO4 + autoDW solution and centrifugation (3000 rpm / 3 min). 10 μl of 20 mM HEPES (ph7.2) + 100 mM NaCl + 2.5 mM EGTA (Reaction buffer) was added to the beads and 4.5 μl of Reaction buffer was added to 0.5 μl of PI, followed by incubation at room temperature for 10 minutes. Thereafter, 5 μl of 20 mM HEPES and 20 μM MnCl 2 + 20 μM ATP + r-32P 0.5 μl + auto DW were added and reacted at 30 ° C. for 20 minutes. 100 μl of 1 M HCl was injected to terminate the reaction. 200μl of CHCl3: CHOH (1: 1) solution was added to the finished beads, and when separated into two layers, only 80μl of the lower layer solution was added, and 80μl of 1M HCl: CH3OH (1: 1) was added. 50 µl of a solution was obtained. The solution was centrifuse (5000 rpm / 30 min) and the solution was vaporized to leave only beads. 10 μl of CHCl 3: CH 3 OH (1: 1) was added thereto, followed by TLC.

(6) Shelf -One kinase assay

After collecting the cells, perform Bio-rad protein measure (300ug / 300µl), then 1% (v / v) NP-40, 10% (v / v) glycerol, 137 mmol / L NaCl, 20 mmol / L Tris- MIX with HCl (pH 7.4), 1% (v / v) Triton X-100, and protease inhibitors. 1 μl of Anti-Raf-1 antibody was added to the substrate, followed by 4 ° C. overnight rotary shaking. 100 μl of protein beads were added to the cell and reacted at 4 ° C. for 2 hours, followed by 20 mmol / L HEPES (pH 7.4), 5 mmol / L MnCl 2, 10 mmol / L MgCl 2, 1 mmol / L DTT, 10 μCi of [ Reaction with γ-32P] ATP and 1 mg / mL MBP substrate was followed by electrophoresis using SDS-polyacrylamide gel and luminescence on the film by autoradiography.

(7) Actived Ras affinity precipitation assay

After collecting the cells, perform Bio-rad protein measure (300ug / 300µl), then 1% (v / v) NP-40, 10% (v / v) glycerol, 137 mmol / L NaCl, 20 mmol / L Tris- MIX was performed with HCl (pH 7.4), 1% (v / v) Triton X-100, and protease inhibitors. 5 μg of Raf-1 RBD agarose beads were taken and reacted at 4 ° C. for 1 hour, followed by washing buffer (25 mM HEPES (pH 7.5), 10 mM MgCl 2, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 wash three times with mM Na3VO4, 10% glycerol, 10 μg / ml leupeptin, 10 μg / ml aprotinin, and 25 mM NaF. After electrophoresis using SDS-polyacrylamide gel, it was confirmed as monoclonal pan-Ras to identify Active Ras (GTP-Ras).

(8) Invasion assay

As a method for confirming the invasiveness of cancer cells, after coating OPTI-MEM and Matrigel on the transwell in each well using 24 trans wells, the cells were put in 1X104 cells / ml, and DMSO was administered to the control group and Eckol was administered to the experimental group. After 24 hours, the infiltrated cells were identified through a microscope (X40).

(9) irradiation

Experimental method to check apoptosis after combined treatment of radiation and radiation + ecol using a radiation irradiator. Irradiation 6Gy (3.81gy / min) for complex treatment After the collection of cells, the cell death was confirmed using FACS.

(10) Animal Experiment

Nude mice were used to inject cancer stem cells into subcutaneous tissue at 2 × 10 4 cells / ml. In addition, DMSO was injected into the control group at three days intervals, and Eckol was injected into the experimental group for 28 days. Tumor size was measured by height x major axis x short axis.

Example  1: against cancer stem cells Ecole  effect

(1) cell death

Eckol used FACS analysis to confirm apoptosis in brain tumor cells, and apoptosis experiments were also performed on brain cancer stem cells.

As a result, as shown in Figure 1, after the treatment of the Eckol (Eckol) by concentration in the cancer stem cells as a result of confirming the effect on the cells, did not have any effect on cell death.

(2) cancer stem cell size

After formation of cancer stem cells with conditioned media and passage twice, cancer stem cells (Spheres) were administered to 50 μm-sized cancer stem cells, DMSO was administered to the control group and Eckol was administered to the experimental group, and 72 hours later, the cells were identified under a microscope (X40). do. Check the cell size using the Motic lmage plus 2.0 program.

As a result of confirming the size of the cancer stem cells after the treatment with the cancer stem cells, it was confirmed that the growth of the cancer stem cells as shown in FIG.

(2) cancer stem cell marker expression

Treatment with Eckol (Eckol) confirmed the cancer stem cell marker CD133 Papulation, and Western Bloting experiments to confirm the protein expression of Nestin, CD133, Musashi-1.

As a result, as shown in FIG. 3, CD133 expression, which is a brain cancer cancer stem cell marker, was compared through FACS analysis. As a result, it was confirmed that CD133 expression was reduced after the treatment. In addition, by comparing the protein expression patterns of brain cancer stem cell markers Nestin, CD133, Musashi-1 using western blot method, it was confirmed that the expression was significantly reduced after the treatment.

In addition, the expression patterns of the cancer stem cell markers were confirmed by immunofluorescence staining.

As a result, as shown in Figure 4, comparing the expression of the stem cell markers Nestin, CD133, Musashi-1, as a result of the expression was confirmed that the expression is suppressed after treatment

(4) Self-renewal

The cancer stem cells were made into single cells, and then cultured with only one cell in a 96 well plate, and then subjected to a self-renwral assay to form cancer stem cells.

The expression of Notch2 and Sox2 proteins involved in self-renewal in cancer stem cells was confirmed by immunofluorescence staining and Western blotting.

In addition, the expression pattern of Tuj-1 used as a differentiation marker (differentiation) was confirmed by the expression of immunofluorescence (immunofluorescence stain) and the expression was changed when the exol treatment.

As a result, as shown in FIG. 5, it was confirmed that the autologous regeneration ability of cancer stem cells was significantly suppressed in the cells treated with the extract. In addition, the expression of Sox2, Nocth2 and β-catenin, which are self-renewal markers, was reduced, and the expression of Sox2 was reduced in cells treated with immunofluorescence stain and Tuj-, a differentiation marker. It was confirmed that the expression of 1 is increased.

Example  2: effect on anticancer drugs Ecole  effect

In parallel with radiation therapy and anticancer drugs, 6Gy was irradiated at a dose rate of 3.81Gy / min as a single radiation and 6Gy was irradiated after FAC analysis. In addition, the experiments with anticancer drugs were treated with 10 μM of Temozolomide as a control group, and treated with Temozolomide and Ecol as experimental groups, and then subjected to FACS analysis for apoptosis after 72 hours.

As a result, as shown in Figure 6, the brain cancer cells and brain cancer stem cells were treated with radiation (6Gy) and anticancer (temozolomide), it was confirmed that apoptosis does not occur in brain cancer stem cells. However, when Eckol was treated with brain cancer stem cells resistant to radiation and anticancer drugs, apoptosis was remarkably increased in brain cancer stem cells that did not cause cell death.

Example  3: through animal testing Tumor formation ability  Confirm

To determine the effect on tumorigenic capacity, tumors were made in immunodeficient transgenic mice. Cancer stem cells were injected into the nude mouse, DMSO was injected into the control group, and 10 mg / kg of Eckol was injected into the experimental group three times every three days, followed by 28 days of experiments. It was confirmed whether the formation was suppressed.

In addition, a soft agar assay was performed to investigate the malignant trait of tumors in the in-vitro.

As a result, as shown in FIG. 7, it was confirmed that tumor formation was suppressed in the immunodeficient transgenic mice treated with exol (FIG. 7A).

In addition, cancer stem cells grew well in the soft agar and showed malignant traits, whereas the treated cancer stem cells did not grow in the soft agar, indicating that the malignant traits of tumors were suppressed (FIG. 7B).

Example  4: invasiveness of cancer stem cells

Malignant tumors are able to metastasize and invasion to the surroundings, and cancer stem cells are also known to be good at metastasis and invasion. In order to confirm the invasive activity of cancer stem cells, the invasion assay was performed to confirm whether Eckol inhibited the invasive activity.

In addition, Western Blotting observed the protein expression of MMP-2 / 9, a protein involved in invasive activity.

As a result, as shown in Figure 8, the result of examining the effect of Eckol (Eckol) on the invasion capacity of brain cancer stem cells, it was found that Eckol inhibits the invasion (cancer) of cancer stem cells.

In addition, it was confirmed that the activity of matrix metalloproteinase (MMP) -2/9 enzyme, which plays an important role in metastasis and invasion of tumor cells, was significantly reduced in Eckol-treated cells. ..

Example  5: Ecole  Signal transduction activity Low performance

The effects of Eckol on PI3Kinase signaling and Ras-Raf-ERK signaling, which are important signaling pathways for tumor cell growth, metastasis and apoptosis inhibition, were investigated.

PI3Kinase signaling activity is known as an important signaling mechanism for maintenance, malignance and invasive activity of cancer stem cells, and confirmed PI3Kinase activity in Eckol-treated cancer stem cells. The degree of inhibition of PI3K kinase activity was compared by the known inhibitors of PI3K (LY294002) and Eckol.

In addition, the effects of exel in Ras-Raf-ERK signaling system were examined through kinase experiments, Raf-1 activity experiments, and Western blotting.

The results are shown in Fig.

First, when the exol was treated, it was found that the kinase activity of the upper signal PI3K was suppressed and the lower signals were suppressed (FIG. 9A), and the same effect that Eckol and Inhibitor (LY294002) of PI3K inhibited the activity of PI3K. It was confirmed that (Fig. 9B). In addition, it was confirmed that Eckol suppresses Ras signal through Ras-Raf-ERK signaling system, thereby suppressing lower signals, thereby inhibiting maintenance and malignant and invasive activity of cancer stem cells (FIG. 9C).

These results suggest that Eckol may be used in combination with existing anticancer agents or radiation therapy for the treatment of brain cancer, thereby inducing the death of cancer stem cells and cancer cells, thereby enabling more effective cancer treatment.

Claims (20)

A pharmaceutical composition for inhibiting cancer stem cell growth, which has the ability to differentiate into brain cancer or glioblastoma, containing an icol compound of formula 1 that inhibits PI3K and Ras signaling activity as an active ingredient:
[Formula 1]

The pharmaceutical composition of claim 1, wherein the eckol compound is derived from brown algae.
According to claim 2, wherein the brown algae are Eisenia bicyclis, Eisenia arborea, Eisenia desmarestioides, Eisenia galapagensis ), Eisenia masonii, Eclonia kurome, Eclonia cava, Eclonia stolonifera, Eclonia maxima, Eclonia radidia Ecklonia radiata, Eclonia bicyclis, Eclonia biruncinate, Eclonia buccinalis, Eclonia caepaestipes, Eclonia ex Ecklonia exasperta, Eclonia fastigiata, Eclonia brevipes, Eclonia arborea, Ecklonia Selection from the group consisting of Ecklonia latifolia, Ecklonia muratii, Ecklonia radicosa, Ecklonia richardiana and Ecklonia wrightii Pharmaceutical composition, characterized in that.
The pharmaceutical composition according to claim 3, wherein the brown alga is Ecklonia cava.
delete delete delete The pharmaceutical composition of claim 1, wherein the composition inhibits Ras signal through a Ras-Raf-ERK signaling system.
According to claim 1, wherein the composition is a pharmaceutical composition, characterized in that administered in combination with the anticancer drug temozolomide (temozolomide). delete delete delete delete delete delete delete delete delete delete delete

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