JP2014221752A - Differentiation promoting agent for cancer stem cell and brain tumor therapeutic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a therapeutic agent through a process of differentiation of a cancer stem cell to an un-cancer stem cell in various tumors.SOLUTION: A differentiation promoting agent contains at least one of metformin or its pharmacologically acceptable salt as an active ingredient and exhibits the action of promoting differentiation of a cancer stem cell to an un-cancer stem cell included in brain tumor.

Description

  The present invention relates to a differentiation promoting agent and a brain tumor therapeutic agent for cancer stem cells.

  Compared with normal somatic cells, cancer cells that make up various types of tumors all have the same number of cell divisions and are highly proliferative, infiltrate surrounding tissues, and metastasize to other parts of the body. It was thought that it had the feature of doing. On the other hand, recent research has shown that a large number of cancer cells that make up a single tumor are diverse, and a small group of cancer cells that function as a part of the cancer function as “cancer stem cells”. It is becoming clear that it has tumorigenicity by having multipotency capable of differentiating into various types of cells. The basic concept of cancer stem cells is based on the concept of irreversibility that non-cancer stem cells generated by differentiation of cancer stem cells have no tumorigenicity and can no longer return to cancer stem cells (Non-Patent Documents). 1).

Based on the above findings, various drugs have been developed to suppress the progression of various tumors and prevent metastasis by killing cancer stem cells or differentiating cancer stem cells into non-cancer stem cells. (For example, Patent Documents 1 to 3).
Patent Documents 1 and 2 describe that a predetermined polyether compound and an acetaminophen derivative reduced cancer stem cells such as breast cancer and lung cancer in a predetermined medium, respectively. Patent Document 3 describes killing cultured breast cancer-derived cancer stem cells and non-cancer stem cells by using metformin, an oral hypoglycemic agent, in combination with doxorubicin, an anticancer agent. Yes.

JP 2011-213612 A JP 2012-31076 A Special table 2013-503171 gazette

Medema, Nat Cell Biol 2013; 15: 338-344

  However, to date, there are only a few reports on therapies targeting cancer stem cells, all of which are limited to preliminary studies in vitro. It has not been. The discovery of drugs that transform cancer stem cells into non-cancer stem cells, etc., makes it possible to suppress the recurrence of tumors in various tumors, delay and extinguish the progression of various tumors, prevent metastasis, etc. Treatment is thought to be promoted. In particular, non-cancer stem cell conversion of cancer stem cells is expected to be a very effective treatment method for tumors that cannot be completely excised by surgical treatment or for tumors where tumor recurrence has a significant prognosis.

  Therefore, the present invention has an object to provide a therapeutic agent through the process of differentiating cancer stem cells into non-cancer stem cells in various tumors. In particular, the present invention is an invasive tumor and the boundary with normal tissue is unclear. Therefore, it is an object of the present invention to provide an effective therapeutic agent for a brain tumor that is difficult to be cured only by a surgical procedure. It is another object of the present invention to provide a therapeutic agent that is effective for primary brain tumors including gliomas such as glioblastoma that progresses very rapidly due to rapid tumor growth.

As a result of detailed studies, the present inventors have found that metformin is useful as a therapeutic agent for brain tumors. In addition to the above, the present inventors have also found various new uses of metformin.
The present invention has been achieved based on such new findings.
The present invention is as follows.
[1] A differentiation-promoting agent that contains at least one of metformin or a pharmacologically acceptable salt thereof as an active ingredient and exhibits an action of promoting the differentiation of cancer stem cells contained in brain tumors into non-cancer stem cells.
[2] The differentiation promoting agent according to [1], wherein the cancer stem cell expresses at least one molecular marker selected from the group consisting of musashi, nestin, bmi1 and sox2.
[3] A therapeutic agent for brain tumors containing at least one of metformin or a pharmacologically acceptable salt thereof as an active ingredient.
[4] The therapeutic agent for brain tumor according to [3], wherein the brain tumor includes cancer stem cells expressing at least one molecular marker selected from the group consisting of musashi, nestin, bmi1 and sox2. .
[5] The therapeutic agent for brain tumor according to [3] or [4], wherein the dose of the active ingredient is 500 mg / day to 3000 mg / day.
[6] The therapeutic agent for brain tumor according to any one of [3] to [5], further comprising a component capable of controlling the blood glucose concentration to 4.44 mmol / L to 6.06 mmol / L.
[7] The therapeutic agent for brain tumor according to any one of [3] to [6], further comprising a glucose metabolism antagonist as an active ingredient.
[8] The brain tumor according to [7], which contains at least one selected from the group consisting of 2-deoxyglucose, 3-bromopyruvate, and 3-bromo-2-oxopropionate-1-propyester as the glucose metabolism antagonist. Remedy.
[9] The therapeutic agent for brain tumor according to any one of [3] to [6], further comprising a chemotherapeutic agent for brain tumor as an active ingredient.
[10] The chemotherapeutic agent contains at least one selected from the group consisting of temozolomide, nimustine, carmustine, lomustine, ranimustine, procarbazine, bevacizumab, cisplatin, carboplatin, vincristine, bleomycin, interferon, methotrexate, cytarabine [9] ] The therapeutic agent of the brain tumor of description.
[11] A method for treating a brain tumor using at least one of metformin or a pharmacologically acceptable salt thereof as an active ingredient.
[12] The brain tumor treatment method according to [11], wherein the brain tumor includes cancer stem cells that express at least one molecular marker selected from the group consisting of musashi, nestin, bmi1 and sox2. .
[13] The brain tumor treatment method according to [11] or [12], wherein the dose of the active ingredient is 500 mg / day to 3000 mg / day.
[14] The method for treating a brain tumor according to any one of [11] to [13], further comprising a component capable of controlling the blood glucose concentration to 4.44 mmol / L to 6.06 mmol / L.
[15] The method for treating a brain tumor according to any one of [11] to [14], further using a glucose metabolism antagonist as an active ingredient.
[16] The brain tumor treatment according to [15], wherein at least one member selected from the group consisting of 2-deoxyglucose, 3-bromopyruvate, and 3-bromo-2-oxopropionate-1-propyester is used as the glucose metabolism antagonist. Method.
[17] The method for treating a brain tumor according to any one of [11] to [14], further using a chemotherapeutic drug for the brain tumor as an active ingredient.
[18] As the chemotherapeutic agent, at least one selected from the group consisting of temozolomide, nimustine, carmustine, lomustine, ranimustine, procarbazine, bevacizumab, cisplatin, carboplatin, vincristine, bleomycin, interferon, methotrexate, cytarabine is used [17]. A method for treating a brain tumor according to claim 1.

  ADVANTAGE OF THE INVENTION According to this invention, while providing the therapeutic agent of the tumor which uses the component conventionally administered to the human body as an active ingredient, the novel therapeutic agent especially about a brain tumor can be provided.

3 is a graph showing the results according to Example 1. 6 is a graph showing the results according to Example 2. 4 is an electrophoresis photograph according to Example 3. It is the graph which showed the result concerning Example 4. 10 is a graph showing the results according to Example 5. 6 is an electrophoresis photograph according to Example 6; It is the graph which showed the result concerning Example 7. FIG. 10 is a graph showing the results according to Example 8. 10 is a graph showing the results according to Example 9. 10 is a graph showing the results according to Example 10. It is the graph which showed the result concerning Example 11. FIG. It is the graph which showed the result concerning Example 12. FIG. It is the graph which showed the result concerning Example 13. FIG.

  The present invention reduces the proliferation ability inherent to cancer stem cells by allowing Metformin, known as a therapeutic agent for diabetes, to act alone on a cultured cell line of cancer stem cells established from surgically isolated tissue. Based on the decrease and disappearance of molecular markers peculiar to cancer, and the fact that a long survival time was secured in mice transplanted with cancer cells treated with metformin compared to mice transplanted with normal cancer stem cells It is. Further, it is based on the fact that the survival period can be extended by administering metformin alone to mice transplanted with cancer stem cells.

In the present specification, “to” indicates a range including numerical values described before and after that as a minimum value and a maximum value, respectively.
In this specification, the amount of each component in the composition means the total amount of the plurality of substances present in the composition unless there is a specific notice when there are a plurality of substances corresponding to each component in the composition. .
The present invention will be described below.

<Metformin>
Metformin is an alternative name for N, N-dimethylimidbonbonide diamond (substance name according to IUPAC nomenclature) (CAS registration number: 657-24-9), and is also called 1,1-dimethylbiguanide and has the chemical formula C ₄ H ₁₁ N ₅ ( The molecular weight is 129.164 g / mol).
Examples of the pharmacologically acceptable salt of metformin include metformin hydrochloride. Metformin hydrochloride represented by the following structure is sold by Sumitomo Dainippon Pharma Co., Ltd. under the trade name Metogluco (registered trademark).

In the present invention, either one of metformin and a pharmacologically acceptable salt thereof may be used alone, or two or more thereof may be used in combination.
In the present specification, metformin and pharmacologically acceptable salts thereof are sometimes collectively referred to as “metformin” hereinafter.

The cancer stem cell targeted in the present invention is an undifferentiated cell having a high tumor forming ability, self-replicating in an undifferentiated state (producing cancer stem cells by cell division), and more It is a cancer cell characterized by its ability to change (differentiate) into a non-cancer stem cell with a high degree of differentiation and no tumorigenicity. As a method for detecting the presence and frequency of cancer stem cells, transplantation experiment methods in which tumor cells are transplanted into experimental animals to observe tumor forming ability are widely used by those skilled in the art.
Moreover, cancer stem cells are proteins involved in the maintenance of various stem cells including embryonic stem cells, and bmi1 (Blymphoma Mo-MLV insertion region 1 homolog) used as a kind of molecular marker, embryonic stem cells It is also a transcription factor involved in the maintenance of various stem cells including the above, and is also characterized by expressing sox2 used as a kind of molecular marker.

  On the other hand, the non-cancer stem cell in the present invention means a cancer cell other than a cancer stem cell which is a cell forming a tumor tissue. Non-cancer stem cells have no tumorigenicity and are characterized by low expression of molecular markers that are specifically expressed by cancer stem cells compared to cancer stem cells.

Differentiation from cancer stem cells to non-cancer stem cells is confirmed by measuring the expression level of molecular markers that are specifically expressed by cancer stem cells and molecular markers that are specifically expressed by non-cancer stem cells by a commonly used method. can do. Specifically, it can be measured by flow cytometry of molecular markers, immunocytochemistry, Western blot analysis, etc. by the method described in Piccilillo, Nature 2006; 444: 761-765. Regarding the cancer stem cells of brain tumors used in the examples of the present invention, expression of differentiation markers such as βIII-tubulin and GFAP that are specifically expressed in non-cancer stem cells generated by differentiation of cancer stem cells of brain tumors, Differentiation can be confirmed.
In addition, as a basic concept of cancer stem cells, it is assumed that change (differentiation) from cancer stem cells to non-cancer stem cells is irreversible (Medema, Nat Cell Biol 2013; 15: 338-344).

As will be clarified in the following examples, a cancer stem cell culture cell line (Matsuda, Sci Rep. 2012) established directly from a surgically removed tissue of glioblastoma (glioblastoma), which is a type of cancer stem cell in brain tumors; 2: 516) was examined for various periods in the presence and absence of metformin. When cultured in the presence of metformin, differentiation of cancer stem cells into non-cancer stem cells was promoted. The result which shows that it is was obtained.
In other words, by culturing cancer stem cells in the presence of metformin, A) the ability to form a suspended cell mass (sphere) in the subsequent culture is reduced, compared with the case of culturing without using metformin, and B) It was confirmed that the expression of cancer stem cell markers decreased and / or disappeared, and C) the expression of various differentiation markers increased. Similarly, when cells cultured for a certain period in the presence and absence of metformin were transplanted into the brain of nude mice, a significantly longer survival time was observed in the group transplanted with cells cultured in the presence of metformin. It was done.
The above results indicate that the ratio (amount) of cancer stem cells is low in the cell group cultured in the presence of metformin, and that metformin promoted differentiation of cancer stem cells into non-cancer stem cells. Can be understood. From this, it is understood that metformin functions as a differentiation promoting agent that promotes differentiation of cancer stem cells into non-cancer stem cells.

The cancer stem cell culture cell line established directly from the surgically removed tissue of glioblastoma used above is an RNA-binding protein that is strongly expressed in neural progenitor cells in addition to bmi1 and sox2, which are molecular markers related to various stem cells. It is an intermediate filament unique to musashi, which is a kind of molecular marker, and progenitor cells (stem cells) of neuroectodermal, and is also characterized by nestin, which is also a kind of molecular marker Metformin is considered to have the same effect on cancer stem cells characterized by the expression of these stem cell markers.
That is, metformin is not limited to cancer stem cells contained in glioblastoma tissues corresponding to gliomas among primary brain tumors, but may be brain tumors that express molecular markers such as bmi1, sox2, musashi, and nestin. For example, it is understood that it functions as a differentiation promoting agent that promotes the differentiation of cancer stem cells into non-cancer stem cells in brain tumors such as metastatic brain tumors.

A brain tumor is an invasive tumor, and its boundary with normal tissue is unclear, so it is said that it is extremely difficult to remove the entire tumor part. Therefore, radiation therapy or chemotherapy is required as a basic treatment for brain tumors after surgery to remove the tumor (Stupp, New England J. Med. 2005; 352: 987-96). In particular, the glioblastoma used for the evaluation in the present invention is a malignant glioma that progresses very rapidly due to rapid tumor growth among brain tumors, and its symptoms worsen in units of several weeks. Among them, it is known that the prognosis is bad, and effective treatment is difficult. In addition, malignant gliomas such as malignant astrocytoma often have common genetic abnormalities with glioblastoma, and there are commonities such as coexistence between tissue types and changes from one to the other. Therefore, the general treatment methods are similar and have similar problems.
The present invention has been found that metformin significantly shows a differentiation promoting action on cancer stem cells derived from glioblastoma having a high malignancy among brain tumors, and the treatment of primary brain tumors such as gliomas. Is expected to contribute.

In addition, the examination in the following examples show that administration of metformin intraperitoneally to nude mice transplanted with cancer stem cells in the brain can significantly extend the subsequent survival period. Metformin has been shown to be effective in the treatment of tumors including it. From this, it is understood that metformin functions as a therapeutic agent for tumors containing cancer stem cells. In this specification, the term “treatment” refers to the effect of delaying the progression of the disease state, extending the survival period, and preventing recurrence after surgery, in addition to the cure of the disease state. Is used to mean
The reason for the survival of nude mice transplanted with cancer stem cells by the administration of metformin is not necessarily clear, but as explained above, it is clear that metformin has an effect of promoting differentiation of cancer stem cells. It is presumed that the differentiation of cancer stem cells into non-cancer stem cells was promoted in the body, resulting in loss of tumor-forming ability and suppression of tumor growth.
It has never been known that metformin acts on cancer stem cells to lose its ability to form tumors, and its systemic administration has a therapeutic effect in animal models of brain tumors. This will lead to the development of new brain tumor therapies that target stem cells. It is also a feature of the present invention that a mechanism for irreversibly losing tumor-forming ability by changing cancer stem cells into non-cancer stem cells does not simply kill cancer stem cells. In the present invention, it was shown that metformin alone has a therapeutic effect targeting cancer stem cells.

  Note that the presence of the blood brain barrier poses a problem when metformin is used particularly for pharmacological treatment of brain tumors. In this regard, it is known that metformin can pass through the blood brain barrier (Labuzek, Pharmacol Rep. 2010 Sep-Oct; 62 (5): 956-65). In this respect, metformin is also expected as a therapeutic agent for brain tumors.

  The dose and number of doses of metformin, or a pharmaceutically acceptable salt thereof, as a therapeutic agent for tumors vary depending on the dosage form, patient age, body weight, nature or severity of symptoms to be treated, etc. It is preferable that the amount is as long as it does not cause a problem. Specifically, from the viewpoint of tumor treatment, it is preferable to administer once or several times a day with an upper limit of about 3000 mg / day. However, it goes without saying that these doses and the number of doses are desirably adjusted according to the various conditions described above.

  As described above, it is preferable that the dosage of metformin as a therapeutic agent for tumors is large as long as side effects do not become a problem. Specifically, 2550 mg / day, which is the upper limit recognized by the US FDA as a therapeutic agent for diabetes, or If it is about 2250 mg / day or less recognized in Japan, it is preferable at the point which can be safely administered without a remarkable side effect. Moreover, in order to produce a substantial medicinal effect as a therapeutic agent for tumors, administration of about 500 mg / day or more is preferable.

  It should be noted that the dose of metformin when the survival effect was observed in nude mice transplanted with the above cancer stem cells was equivalent to about 2500 mg / day in terms of human (body weight 60 kg). In the US FDA, the amount used as an antidiabetic agent is 2550 mg / day, the amount used in Japan is 2250 mg / day, and administration within a range in which side effects are sufficiently suppressed even by single administration of metformin Therefore, a therapeutic effect on the tumor is expected.

  The method of administration of metformin is arbitrary. For example, not only oral administration but parenteral administration may be used. Examples of parenteral administration include subcutaneous injection, intravenous injection, intramuscular injection, and intraperitoneal injection.

  When metformin is administered orally, it is used in the form of tablets, capsules, powders, solutions, elixirs, etc., and when administered parenterally, it is used in a sterilized liquid form such as solution or suspending agent.

When metformin is used in the form as described above, a solid or liquid non-toxic pharmaceutical carrier may be included in the composition.
As an example of the solid carrier, a normal gelatin type capsule is used.
These capsules, tablets and powders generally contain 5% to 95% by weight, preferably 5% to 90% by weight, of the active ingredient relative to the total weight of the preparation.
As the liquid carrier, water, petroleum, peanut oil, soybean oil, mineral oil, sesame oil, physiological saline, dextrol, similar sucrose solution, ethylene glycol, propylene glycol, polyethylene glycol and the like can be used.
As previously discussed, the effect of metformin as a tumor therapeutic agent is presumed to be due to metformin's ability to promote the differentiation of cancer stem cells, resulting in decreased or lost tumor-forming ability in tumor tissue It is guessed that. On the other hand, as shown in the results of Examples, metformin is considered not to have an active killing effect on cancer cells. For this reason, as a treatment method using metformin, after removal of the tumor by surgical treatment, administration of metformin is performed with a small amount of residual tumor, and recurrence or metastasis of macroscopic tumor tissue due to the residual tumor. It is considered preferable to adopt a usage whose main purpose is prevention.

Further, from the examination in the following examples, when metformin promotes differentiation of cancer stem cells into non-cancer stem cells, the concentration of glucose present in the cell culture environment affects, and the promotion of differentiation is enhanced by the decrease in glucose concentration. It has become clear. Although the mechanism that causes such a phenomenon is not always clear, when metformin is administered to the human body as a tumor therapeutic agent, the effect of metformin as a tumor therapeutic agent is increased by reducing the glucose concentration in the body by appropriate means. Be expected.
It is well known that various stresses associated with various diseases including cancer will lead to a hyperglycemic condition in the future (Nomicos, J Clin Med Res. 2012 Aug; 4 (4): 237-41). The possibility that the hyperglycemic state may inhibit the therapeutic effect of metformin on cancer stem cells is well suspected. Metformin itself is an anti-diabetic drug, so it is expected to have a hypoglycemic effect. By inhibiting uptake and creating a hypoglycemic state, it is possible to enhance the therapeutic effect of metformin on cancer stem cells. The present invention includes, as a part thereof, a novel therapeutic method for cancer stem cells, which is a combination of a therapeutic agent targeting such glucose metabolism and metformin.

  It is desirable that the glucose concentration in the body when metformin is administered to the human body as a tumor therapeutic agent is low within a range that does not adversely affect the human body, and the blood glucose concentration is 4.44 mmol / L to 6.06 mmol / L (80 mg / L). dL to 109 mg / dL).

By controlling the blood glucose concentration to 4.44 mmol / L to 6.06 mmol / L (80 mg / dL to 109 mg / dL), compared with the case where the blood glucose concentration is 110 mg / dL or more, It is expected that the therapeutic effect targeting cancer stem cells will be enhanced.
In particular, the blood glucose concentration is more preferably controlled to be 4.44 mmol / L to 5.22 mmol / L (80 mg / dL to 94 mg / dL).
Here, in this specification, blood glucose concentration means fasting blood glucose concentration. In addition, what is necessary is just to measure a blood glucose level according to a normal method after blood collection.

As for the glucose concentration in the body when metformin is administered to the human body as a therapeutic agent for tumors, in addition to dietary restrictions and the like, a drug having an effect of lowering the glucose concentration can be used in combination with metformin.
In addition, it is more preferable to use a glucose metabolism antagonist in combination with metformin. This is expected to synergistically improve the therapeutic effect of tumor therapeutic agents targeting cancer stem cells.
The glucose metabolism antagonist is not particularly limited as long as it is a component that suppresses glucose metabolism in the body. Specific examples of the glucose metabolism antagonist include 2-deoxyglucose, 3-bromopyruvate, 3-bromo-2-oxopropionate-1-propylester, and the like.

  The administration method of metformin and an antimetabolite is arbitrary. For example, not only oral administration but parenteral administration may be used. Examples of parenteral administration include subcutaneous injection, intravenous injection, intramuscular injection, and intraperitoneal injection.

In the present invention, the first selection for malignant gliomas such as the glioblastoma is performed on nude mice transplanted in the brain with cancer stem cells established from the tissue of glioblastoma as described above. The combined use of temozolomide, which is used as a chemotherapeutic agent, with metformin further extends the survival period from transplantation of cancer stem cells to brain tumors when nude mice die of brain tumors, and metformin alone and other chemicals such as temozolomide It was shown that the therapeutic effect can be exerted even in combination with therapeutic drugs.
Thus, in the present invention, it has been shown that metformin alone has a therapeutic effect targeting cancer stem cells, and that combination therapy with other anticancer agents such as chemotherapeutic drugs is effective. The present invention includes the treatment of brain tumors such as glioma by using metformin alone and in combination with other anticancer agents.

The reason why the combined use of metformin and chemotherapeutic drugs increases the therapeutic effect on nude mice transplanted with cancer stem cells in the brain is not necessarily clear, but metformin promotes differentiation of cancer stem cells and kills non-cancer stem cells with chemotherapeutic drugs Are considered to be synergistic mechanisms, and the presence of metformin changes the sensitivity of cancer stem cells and non-cancer stem cells to chemotherapeutic drugs.
The chemotherapeutic agent used in combination with metformin is not particularly limited as long as the therapeutic effect of the tumor to be treated is expected. As chemotherapeutic drugs used in combination with metformin, specifically, in the case of glioma, in addition to temozolomide, alkylating agents such as nimustine, carmustine, lomustine, ranimustine, procarbazine generally used for brain tumors, etc. Molecular target drugs such as bevacizumab, platinum preparations such as cisplatin and carboplatin, bleomycin, interferon, methotrexate / cytarabine, and vincristine.
The dose of the chemotherapeutic drug used in combination with metformin can be appropriately set according to the type of chemotherapeutic drug to be used, but it is desirable to adjust appropriately in consideration of the action caused by the combined use with metformin. .
In addition, since metformin is thought to promote the differentiation of cancer stem cells into non-cancer stem cells, in addition to the combined administration of the above chemotherapeutic drugs, it is particularly intended for killing non-cancer stem cells such as radiotherapy. The combined use with the conventional cancer therapy is considered effective. Furthermore, it is considered effective to use metformin in combination with other differentiation promoting agents that promote the differentiation of cancer stem cells into non-cancer stem cells.
In addition, by utilizing the fact that metformin promotes the differentiation of cancer stem cells into non-cancer stem cells and has no significant side effects, tumors that are difficult to cure, especially only by surgical treatment such as brain tumors, etc. To prevent recurrence after these treatments for brain tumors that are difficult to cure only by surgical treatment, radiation therapy, chemotherapy, or other conventional cancer treatments It is also effective to administer an appropriate amount.

  Further, the therapeutic agent for brain tumor of the present invention is not limited to humans, for example, for warm blooded animals such as mice, rats, rabbits, sheep, pigs, cows, horses, birds, cats, dogs, monkeys, chimpanzees and the like. It is valid.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to them.
[Example 1]
In order to examine the effect of metformin on the promotion of differentiation of cancer stem cells in brain tumors, in particular, a cancer stem cell culture cell line established directly from the surgically isolated tissue of glioma, which is representative of primary brain tumors, among which the most aggressive glioblastoma (Matsuda, Sci Rep. 2012; 2: 516) was used for the following investigation.
One of the important features of cancer stem cells derived from brain tumors is the formation of floating cell mass (Dirks, Mol Oncol. 2010 Oct; 4) without adhering to the culture dish under normal stem cell culture conditions. (5): 420-30). Therefore, in the following examination, the effect of metformin on the differentiation of cancer stem cells was examined using this property as an index.

Matsuda, Sci Rep. 2012; 2: 516, cancer stem cells established directly from glioblastoma surgically-extracted tissue are cultured in a medium containing 1 mM metformin (Metformin) and a medium not containing (Control) for 3 days, and a medium not containing metformin After washing and removing metformin using 1, 1 × 10 ³ cells were collected from the cells under each culture condition and cultured under the sphere formation conditions (conditions described in Matsuda, Sci Rep. 2012; 2: 516). The medium was adjusted to 26.2 mM by adding glucose to a commercially available culture solution having a glucose concentration of 17.5 mM (DMEM / F12 medium sold by Invitrogen, product number 17504-044). .
The results are shown in FIG. In FIG. 1, “1st passage” indicates the number of spheres after 3 days of sphere formation culture. Three days after the start of sphere formation culture, the cells were separated, and the number of spheres after culturing 1 × 10 ³ cells again under sphere formation conditions for 3 days was counted as the sphere number of “2nd passage”. In the figure, “*” indicates that there is a statistically significant difference (P <0.05).
As is clear from the results shown in FIG. 1, even after the removal of metformin by washing, the cells cultured in a medium containing metformin continuously suppress the sphere-forming ability of cancer stem cells. Has been. That is, it was revealed that metformin suppresses sphere formation by cancer stem cells. This result indicates that metformin may induce cancer stem cells to differentiate into non-cancer stem cells, may significantly suppress the growth of cancer stem cells, and may also kill cells.

[Example 2]
The presence or absence of cell killing by metformin was examined.
When cancer stem cells established directly from glioblastoma surgically-extracted tissue by the same method as described in Example 1 are cultured in a medium with a glucose concentration of 26.2 mM for 3 days, and a medium not containing metformin, and A medium containing metformin at each concentration of 1 mM to 10 mM was used, and the number of living cells and the number of dead cells after culture were counted. Cell viability was determined by the dye exclusion method, and cell viability was calculated by viable cell count / (viable cell count + dead cell count).
FIG. 2 shows the results in a medium with metformin concentrations of 0 mM, 1 mM, and 10 mM. According to this study, when the metformin concentration is 6 mM or less, the cell viability is not decreased, and as is clear from the results shown in FIG. 2, the metformin concentration (1 mM) employed in Example 1 is used. No decrease in cell viability was observed.
Considering this result, the result of Example 1 was not caused by cell killing by metformin, but cancer stem cells were induced to differentiate into non-cancer stem cells and / or proliferation of cancer stem cells was remarkably suppressed. Inferred to be the result.

[Example 3]
The degree of differentiation of cancer stem cells after culturing cancer stem cells in the presence of metformin was examined.
Brain stem cancer stem cells generally form spheres, but brain tumor cells that form spheres are not necessarily cancer stem cells. Therefore, the expression of a cancer stem cell marker (Dahlot, Int J Clin Exp Pathol. 2013; 6 (3): 334-48) in the cancer stem cells used in the experiment was examined.
After culturing cancer stem cells directly established from glioblastoma surgically-extracted tissue by a method similar to that described in Example 1 in a medium containing 1 mM metformin (Met) and a medium not containing (Cont.) For 3 days, It was collected. Matsuda, Sci Rep. 2012; 2: 516, proteins in the cells were eluted, and the expression levels of various proteins (Nestin, Musashi, Bmi1, GFAP, βIII-tubulin, Actin) were analyzed by Western blotting. In addition, the expression level of Actin was analyzed in order to confirm that an equal amount of protein was migrated.
The results are shown in FIG. As apparent from the results shown in FIG. 3, glioblastoma cancer stem cells cultured in a medium not containing metformin (Cont. Lane in FIG. 3) express stem cell markers such as Nestin, Bmi1, and Musashi. It was confirmed that In contrast, the expression of these stem cell markers was reduced or disappeared in cancer stem cells cultured in a medium containing metformin (Met lane in FIG. 3) (Met lane in FIG. 3). In addition, when cancer stem cells are cultured in a medium containing metformin, a differentiation marker for nerve cells (βIII-tubulin), a differentiation marker for astrocytes (glial fibrous acid protein; GFAP) (Singh, Cancer Res. 2003 Sep 15; 63 (18): 5821-8) was expressed.

[Example 4]
Furthermore, the influence on the expression of neuronal differentiation marker (βIII-tubulin) and astrocyte differentiation marker (GFAP) by culturing in a medium containing metformin was examined by immunocytochemistry.
Cancer stem cells directly established from glioblastoma surgically-extracted tissue by the same method as described in Example 1 were cultured in a medium containing 1 mM metformin (Metformin) and a medium not containing (Control) for 3 days and 6 days. After fixation, immunostaining of GFAP and βIII-tubulin was performed by immunocytochemistry. At the same time, the cell nuclei were stained by Hoechst staining to count the total number of stained cells.
The results are shown in FIG. FIG. 4 shows the ratio of GFAP and βIII-tubulin positive cells to the total number of cells. In the figure, “*” indicates that there is a statistically significant difference (P <0.05).
As is clear from the results in FIG. 4, the expression of both βIII-tubulin and GFAP differentiation markers was increased by culturing in a medium containing metformin, and the same tendency as the results in Example 3 was confirmed.
The results of Examples 3 and 4 all indicate that metformin has transformed originally undifferentiated cancer stem cells into differentiated cancer cells, that is, non-cancer stem cells. For this reason, suppression of sphere formation by cancer stem cells by culturing in a medium containing metformin confirmed in Example 1 is considered to be due to the loss of sphere formation ability as a result of differentiation of cancer stem cells into non-cancer stem cells by metformin. Is done.

[Example 5]
In Examples 1 to 4 above, it was speculated that cancer stem cells lost the ability to form sphere due to differentiation induction by metformin. On the other hand, as described above, cancer stem cells are undifferentiated and have the ability to form tumors. Thus, in this example, whether or not cancer stem cells actually lost the tumor-forming ability, which is an important property, was examined by differentiation induction with metformin. In the basic concept of cancer stem cells, it is assumed that the change from cancer stem cells to non-cancer stem cells is irreversible.
Specifically, cancer stem cells cultured in a medium containing metformin were transplanted into a mouse brain, and the tumor-forming ability of the transplanted cancer cells was examined by changing the survival period thereafter.
Cancer stem cells established directly from glioblastoma surgically-extracted tissue by the same method as described in Example 1 were cultured for 3 days in a medium containing 1 mM metformin (Metformin) and a medium not containing (Control), and then metformin After washing, 1 × 10 ⁴ cells were transplanted into the brain of nude mice (5 mice per group), and the survival time until the nude mice died of brain tumor was expressed by Kaplan-Meier survival curve. Various studies using nude mice described in this specification were conducted with the approval of the Yamagata University Animal Experiment Committee.
The results are shown in FIG. In the figure, “*” indicates that there is a statistically significant difference (P <0.05).
As is clear from the results shown in FIG. 5, the mice were transplanted with cancer stem cells cultured in a medium containing metformin as compared with transplanting cancer stem cells cultured in a medium not containing metformin. It was shown that the period until cell death after cell transplantation, that is, the survival time was prolonged.
In addition, as shown in Example 2, it is clear that the concentration of metformin acted in this example is not a concentration that induces cell death. Moreover, metformin exposure is temporary and metformin is removed at the time of transplantation. Taking these into account, the results obtained in Example 5 show that transient contact with metformin irreversibly changed many cancer stem cells into non-cancer stem cells without tumorigenic potential. This idea is supported. That is, it is understood that cancer stem cells differentiate into non-cancer stem cells by culturing in a medium containing metformin, resulting in loss of sphere formation ability under normal stem cell culture conditions and loss of tumor formation ability in vivo.

[Example 6]
In this example, the effect of glucose concentration when metformin is applied is examined with respect to the differentiation promoting action of cancer stem cells exhibited by metformin examined above. That is, in Examples 1 to 5, the culture solution having a glucose concentration of 26.2 mM was used, whereas the effect of metformin at a lower glucose concentration was verified. The culture solution having this glucose concentration is prepared by adding glucose to a commercially available culture solution having a glucose concentration of 17.5 mM (DMEM / F12 medium sold by Invitrogen, product number 11320-082).
Cancer stem cells established directly from glioblastoma surgically-extracted tissues by the same method as described in Example 1 were cultured in a medium containing 1 mM metformin (Metformin +) and a medium not containing (Metformin−). At that time, as a culture solution in each case, a commercially available culture solution having a glucose concentration of 17.5 mM (DMEM / F12 medium sold by Invitrogen, product number 11320-082), and glucose in the culture solution. Cultivation was performed using two types of culture solutions added to a glucose concentration of 26.2 mM. Cells were collected after 3 days of culture, the proteins in the cells were eluted, and the expression levels of various proteins (βIII-tubulin, GFAP, Musashi, Sox2, Actin) were analyzed by Western blotting. The expression level of Actin was analyzed in order to confirm that an equal amount of protein was migrated.
The results are shown in FIG. As is clear from the results shown in FIG. 6, the degree of decrease in the expression of the stem cell marker (Musashi, Sox2) and differentiation under the condition of the glucose concentration of 17.5 mM as compared with the case of the glucose concentration of 26.2 mM. The degree of enhanced expression of the marker (βIII-tubulin, GFAP) was enhanced together. This result shows that the degree of differentiation of cancer stem cells into non-cancer stem cells by culturing in a medium containing metformin is affected by the glucose concentration in the medium, and the promotion of differentiation is enhanced when the glucose concentration is low. It became.

[Example 7]
In this example, the presence or absence of metformin and the effect of glucose concentration were examined by immunocytochemistry for the proportion of non-cancer stem cells after culturing in a cancer stem cell medium.
Cancer stem cells established directly from glioblastoma surgically-extracted tissue by the same method as described in Example 6 were cultured in a medium containing 1 mM metformin (Metformin +) and a medium not containing (Metformin−). At that time, the glucose concentration in the medium was 26.2 mM and 17.5 mM for each case. In each case, the cells were fixed after 3 days of culture, and immunostaining of βIII-tubulin and glial fibrous acid protein (GFAP) was performed by immunocytochemistry. At the same time, the cell nuclei were stained by Hoechst staining to count the total number of stained cells.
The results are shown in FIG. The figure shows the ratio of GFAP and βIII-tubulin positive cells to the total number of cells. In the figure, “*” indicates that there is a statistically significant difference (P <0.05). “Ns” indicates that there is no statistically significant difference.
As is clear from the results shown in FIG. 7, in the medium not containing metformin, the ratio of non-cancer stem cells was about 10% regardless of the glucose concentration. In contrast, in a medium containing metformin, about 40% of cancer stem cells change to non-cancer stem cells at a glucose concentration of 26.5 mM, and more than 80% of cells become non-cancer at this time at a glucose concentration of 17.5 mM. It was suggested that it changed into stem cells.
From this result, it is understood that when metformin promotes differentiation of cancer stem cells into non-cancer stem cells, the glucose concentration present in the cell culture environment is affected, and the differentiation promotion is enhanced at a low glucose concentration.

[Example 8]
From the results of each of the above examples, metformin decreases the sphere-forming ability of a cell group containing cancer stem cells, and the action is caused by metformin promoting differentiation of cancer stem cells into non-cancer stem cells. Became clear. In this example, a test was conducted to reduce the sphere-forming ability of metformin, which is shown over a long period of time on a cell group containing cancer stem cells.
Cancer stem cells established directly from glioblastoma surgically-extracted tissue by the same method as described in Example 6 were cultured in a medium containing 1 mM metformin (Metformin +) and a medium not containing (Metformin−). At that time, the glucose concentration in the medium was 26.2 mM and 17.5 mM for each case. In each case, metformin was washed away after 3 days of culture, and 1 × 10 ³ cells were used for the culture test under sphere formation conditions.
In FIG. 8, “1st passage” indicates the number of spheres after sphere formation culture is performed for 3 days using the above 1 × 10 ³ cells. Furthermore, “2nd passage” indicates the number of spheres after 1 × 10 ³ cells were separated from the medium in which the number of spheres was measured by the “1st passage” and cultured again under sphere formation conditions for 3 days. Hereinafter, the number of spheres was also measured in the same way for “3rd passage” and “4th passage”. In addition, in each culture test after removing metformin, the glucose concentration was unified at 26.2 mM. In the figure, “*” indicates that there is a statistically significant difference (P <0.05), and “ns” indicates that there is no statistically significant difference.
As is clear from the results shown in FIG. 8, when the glucose concentration of the medium containing metformin is 26.2 mM, a clear effect of reducing the sphere formation ability is observed with metformin, but when the sphere formation experiment is repeated There is a tendency to recover the sphere-forming ability once suppressed by metformin. On the other hand, when the glucose concentration was 17.5 mM, it was confirmed that the sphere formation inhibitory effect of metformin persists even if the sphere formation experiment is repeated.
All the results shown in Examples 6 to 8 support the idea that the efficiency of metformin to stably change cancer stem cells into non-cancer stem cells is greatly influenced by glucose concentration. In addition, in a cell group including cancer stem cells exposed to metformin in an environment suitable for metformin exposure such as a low glucose concentration environment, it is difficult to recover the original sphere-forming ability afterwards. This is considered to indicate the possibility of effectively suppressing tumor growth with a burden.

[Example 9]
In Examples 6 to 8 above, it was shown that the glucose concentration at the time of metformin exposure was related to the decrease in the ability to form sphere due to the differentiation induction of cancer stem cells by metformin. Therefore, in this example, whether or not the glucose concentration in the culture medium when cancer stem cells were exposed to metformin had an effect on the suppression of tumor-forming ability of cancer stem cells in the living body was examined. .
Cancer stem cells established directly from glioblastoma surgically-extracted tissue by the same method as described in Example 6 were cultured in a medium containing 1 mM metformin (Metformin +) and a medium not containing (Metformin−). At that time, the glucose concentration in the medium was 26.2 mM and 17.5 mM for each case. Metformin was washed and removed after 3 days of culture, and 1 × 10 ⁴ cells contained in each medium were transplanted into the nude mouse brain (5 mice per group), and the survival time until the nude mouse died of brain tumor. Was represented by a Kaplan-Meier survival curve.
The results are shown in FIG. In the figure, “*” indicates that there is a statistically significant difference (P <0.05).
As is clear from the results shown in FIG. 9, when not exposed to metformin, the survival time was about 40 days regardless of the glucose concentration, whereas when exposed to metformin, the glucose concentration was 26 In the case of 2 mM, the survival period was extended to about 50 days. Furthermore, when the glucose concentration was 17.5 mM, it was extended to about 70 days.
This result shows that the effect of metformin, which reduces or loses the ability to form tumors by changing cancer stem cells to non-cancer stem cells, is enhanced by a decrease in glucose concentration in the cell culture environment. 8 supports the results of the study on the sphere-forming ability in No. 8.

[Example 10]
In the above Examples, it was shown that metformin differentiates cancer stem cells into non-cancer stem cells in the medium, and as a result, the sphere-forming ability and tumor-forming ability exhibited by cancer stem cells are reduced or lost. In this example, from the viewpoint of actual treatment, it was examined whether metformin administered systemically could have the therapeutic effect expected from the above example in vivo.
As can be seen from the fact that cancer stem cells were originally defined as cells that can form tumors by transplantation, the frequency of cancer stem cells present in tumors formed in living bodies can be estimated reliably. The only widely recognized method is tumor reimplantation experiments (Nguyen et al. Nat Rev Cancer. 2012 Jan 12; 12 (2): 133-43).

Therefore, the effect of metformin was next verified by a tumor replantation experiment.
First, in order to be able to confirm the formation of tumor under visual observation, cancer stem cells were transplanted subcutaneously into mice, and after the formation of tumor was confirmed, metformin was systemically administered for 10 days. After the administration of metformin for 10 days, the subcutaneous tumor was removed and re-implanted into the brain of another mouse while changing the number of isolated tumor cells.
Cancer stem cells and non-cancer stem cells are present in subcutaneous tumors. When cancer stem cells are included in the re-transplanted tumor cells, a brain tumor is formed, and the mouse dies. Therefore, after the re-transplantation of the tumor cells into the mouse brain, the survival period until the mouse died of the brain tumor was examined.

Cancer stem cells established directly from glioblastoma surgically-extracted tissue by the same method as described in Example 1 were transplanted subcutaneously into nude mice, and when a tumor was formed, the metformin treatment group (Metformin) and the subject group (Control) ).
In the metformin treatment group, 500 mg / kg metformin was intraperitoneally administered once a day.
In the control group, phosphate buffered saline used as a solvent was intraperitoneally administered once a day.
After administration for 10 days in both groups, the subcutaneous tumor was excised the day after the final administration and the tumor cells were separated.
Thereafter, 1,000, 10,000, and 100,000 separated cells (No. of cells translated) were transplanted into the brain of a separately prepared nude mouse (use 5 mice for each group). The results are shown in Table 1. Table 1 shows the number of surviving nude mice in each group and the median survival time (Median) at 160 days after intracerebral transplantation.

In addition, for each group in which 1 × 10 ³ separated tumor cells of the metformin treatment group and the target group were transplanted into the nude mouse brain, the survival time until the nude mouse died of the brain tumor was expressed by a Kaplan-Meier survival curve. . The results are shown in FIG.

As shown in Table 1 and FIG. 10, when subcutaneous tumor cells of mice that were not systemically administered with metformin were transplanted as a control group, brain tumors were reliably formed even by re-implantation of 10 ³ tumor cells, and the mice died. . In contrast, when re-implanted tumor cells derived from subcutaneous tumors of metformin systemic administration group, 10 5 individuals in 2 individuals even ^four to transplanting, 10 ³ 3 individuals in 5 individuals when transplanted to Brain tumor formation was not observed and survival during the study period was confirmed.
These results indicate that systemic administration of metformin reduced the proportion of cancer stem cells present in subcutaneous tumors, and that the effect of metformin administration for a limited period of 10 days was not reversible, and the ability of cancer stem cells to form tumors This proves that metformin can act on cancer stem cells in vivo and produce a therapeutic effect. In particular, in the group in which about 10,000 tumor cells derived from the subcutaneous tumor of the metformin systemic administration group were re-implanted into the brain, there was a significant presence of solids in which no brain tumor formation was observed thereafter. It is inferred that this was the result of almost no cancer stem cells in the tumor cells. That is, it is speculated that the above results show that cancer stem cells were significantly reduced by differentiation or the like in subcutaneous tumors caused by transplantation of cancer stem cells by systemic administration of metformin alone in mice.

[Example 11]
In order to verify the effect of systemic administration of metformin on subcutaneous tumors, in this example, metformin was administered for a short period (10 days) to nude mice transplanted with cancer stem cells subcutaneously by the method described in Example 10. The volume change of the subcutaneous tumor before and after administration was examined.
Cancer stem cells established directly from glioblastoma surgically-extracted tissue by the same method as described in Example 1 were transplanted subcutaneously into nude mice, and when a tumor was formed, the metformin treatment group (Metformin) and the subject group (Control) ).
In the metformin treatment group, 500 mg / kg metformin was intraperitoneally administered once a day. In the control group, phosphate buffered saline used as a solvent was intraperitoneally administered once a day. Administration was carried out for 10 days. Metformin therapy start the (Pre-Treatment) and end (Post-Treatment) tumor volume ([maximum diameter] x [shortest ^diameter] 2/2) was measured.
The results are shown in FIG. As is clear from the results shown in FIG. 11, in the short-term test for 10 days, it was observed that the tumor volume increased about 3 times in the metformin treatment group. Although the degree of increase was small compared to the control group, the difference was slight. From this, the significant life-prolonging effect of metformin administration observed in Example 10 is not a reduction in tumor volume due to direct killing of cancer cells, but a long-term viewpoint due to a decrease in the proportion of cancer stem cells in the tumor. It is presumed that the increase in tumor volume is suppressed.

[Example 12]
In Example 10, in a nude mouse in which a tumor caused by a brain tumor is formed subcutaneously, a specific change occurs in the tumor tissue by systemic administration of metformin, and the progression of the brain tumor that is transplanted again into the brain can be suppressed. It was revealed that metformin has a certain therapeutic effect on the tumor tissue.
However, in order to confirm the applicability of metformin as a therapeutic agent for brain tumors in particular, in addition to the ability of metformin to cross the blood brain barrier, the concentration and duration sufficient for metformin to actually exert a therapeutic effect Should be shown to be able to act on tumor cells in brain tissue.

In addition, even though cancer stem cells are inherent in cancer stem cells at the time of transplantation, tumors are formed by generating not only cancer stem cells but also many non-cancer stem cells by cell division in the process of tumor formation (Reya, Nature). 2001 Nov 1; 414 (6859): 105-11) Therefore, in order to confirm the effect of metformin on cancer stem cells, it is preferable to administer metformin to a group of cells more likely to be cancer stem cells. .
Therefore, in this example, the therapeutic effect of metformin administered systemically was examined on brain tumor formation by cancer stem cells immediately after transplantation into the brain parenchyma.
Therefore, in this example, in order to examine the effect of metformin administered systemically on the formation of brain tumors by cancer stem cells present in brain tissue, metformin immediately after transplanting cancer stem cells into the nude mouse brain. Systemic administration was started, and after the administration was stopped for 5 to 10 days, the survival period until the nude mouse died of brain tumor was examined.
Specifically, the survival period was examined by the following method.
That is, cancer stem cells (1 × 10 ⁴ cells) directly established from glioblastoma surgically-extracted tissue by the same method as described in Example 1 were transplanted into the nude mouse brain, and the subject group (Control), metformin 5 days treatment The treatment was started on the next day after transplantation by dividing into 3 groups (5 mice for each group): a group (Metformin, 5 days) and a metformin 10-day treatment group (Metformin, 10 days).
In the metformin treatment group, 500 mg / kg metformin was intraperitoneally administered once a day for 5 days or 10 days. In the control group, phosphate buffered saline used as a solvent was intraperitoneally administered once a day for 10 days. The survival period from the transplantation of cancer stem cells into the brain to the death of the brain tumor was expressed by a Kaplan-Meier survival curve. The results are shown in FIG. In the figure, “*” indicates that there is a statistically significant difference (P <0.05), and “**” indicates that there is a statistically significant difference (P <0.01).

  As is apparent from the results shown in FIG. 12, nude mice in the untreated group die of brain tumor within 20-30 days, whereas about 5 weeks in the 5-day treated group and about 1 in the 10-day treated group. Month extension of life was shown. This result clearly demonstrates that metformin administered systemically has an effect of suppressing tumor-forming ability against cancer stem cells existing in the brain parenchyma and can exert a therapeutic effect, and shows that it acts as a therapeutic agent for brain tumors. Yes.

[Example 13]
The results obtained in Examples 6 to 12 show that metformin is useful for treatment targeting cancer stem cells alone and particularly effective for treatment of brain tumors.
However, in actual clinical situations, it is assumed that a combination with other therapies is necessary, particularly a situation where there is a residual tumor and it is necessary to use a cytotoxic chemotherapy or the like. For this reason, information about the effectiveness of metformin as a therapeutic agent targeting cancer stem cells is useful when used in combination with such other therapies.

Therefore, in this example, metformin is shown as a therapeutic agent when used in combination with temozolomide, which is a first-line chemotherapeutic agent for malignant gliomas such as glioblastoma used in the examination of the effectiveness of metformin. The usefulness was examined. Here, assuming that temozolomide administration is particularly necessary, that is, initial treatment or additional treatment for residual tumor, unlike Example 12, transplanting cancer stem cells into the nude mouse brain for 10 days. Then, after the tumor was formed, the survival time until the nude mouse died of the brain tumor after various administrations was examined.
Specifically, the survival period was examined by the following method.
That is, cancer stem cells (1 × 10 ⁴ cells) established directly from glioblastoma surgically-extracted tissue by the same method as described in Example 1 were transplanted into the nude mouse brain, and the subject group (Vehicle) and temozolomide monotherapy group (TMZ), metformin monotherapy group (Metformin), and temozolomide and metformin combination treatment group (Met + TMZ) were divided into 4 groups (5 animals in each group), and therapeutic intervention was started 10 days after transplantation.
In the control group, phosphate buffered saline as a solvent was administered intraperitoneally once a day for 15 days, and in the temozolomide monotherapy group, temozolomide 50 mg / kg was administered once a day for 5 days, followed by phosphate buffered saline. 10 days intraperitoneally, metformin treatment group metformin 500 mg / kg once daily for 10 days, followed by phosphate buffered saline intraperitoneally for 5 days, combination treatment group metformin 500 mg / kg for 10 days Temozolomide 50 mg / kg was administered intraperitoneally for 5 days.
The results are shown in FIG. The result of FIG. 13 represents the survival time from the transplantation of cancer stem cells in the brain to the death of the brain tumor in the nude mouse by a Kaplan-Meier survival curve.
As is clear from the results shown in FIG. 13, the high survival rate of the combination treatment group was statistically significant compared to any of the temozolomide monotherapy group, metformin monotherapy group, and the control group. (P <0.05). This finding indicates that metformin can exert its therapeutic effect not only alone but also in combination with temozolomide.

Claims (10)

  A differentiation promoting agent containing at least one of metformin or a pharmacologically acceptable salt thereof as an active ingredient and exhibiting an action of promoting differentiation of cancer stem cells contained in brain tumors into non-cancer stem cells.   The differentiation promoting agent according to claim 1, wherein the cancer stem cell expresses at least one molecular marker selected from the group consisting of musashi, nestin, bmi1 and sox2.   A therapeutic agent for brain tumor comprising metformin or at least one of its pharmacologically acceptable salts as an active ingredient.   The therapeutic agent for brain tumor according to claim 3, wherein the brain tumor includes cancer stem cells expressing at least one molecular marker selected from the group consisting of musashi, nestin, bmi1 and sox2.   The therapeutic agent for brain tumor according to claim 3 or 4, wherein the dose of the active ingredient is 500 mg / day to 3000 mg / day.   Furthermore, the therapeutic agent of the brain tumor of any one of Claims 3-5 containing the component which can control blood glucose level to 4.44 mmol / L-6.06 mmol / L.   The therapeutic agent for brain tumor according to any one of claims 3 to 6, further comprising a glucose metabolism antagonist as an active ingredient.   The therapeutic agent for brain tumor according to claim 7, comprising at least one selected from the group consisting of 2-deoxyglucose, 3-bromopyruvate, and 3-bromo-2-oxopropionate-1-propyester as the glucose metabolism antagonist.   Furthermore, the therapeutic agent of the brain tumor of any one of Claims 3-6 which contains the chemotherapeutic agent with respect to a brain tumor as an active ingredient.   The chemotherapeutic agent contains at least one selected from the group consisting of temozolomide, nimustine, carmustine, lomustine, ranimustine, procarbazine, bevacizumab, cisplatin, carboplatin, vincristine, bleomycin, interferon, methotrexate, cytarabine. For treating brain tumors.