Composition for cancer treatment

Abstract

To provide a composition effective for cancer treatment.SOLUTION: A composition contains plasmalogen extracted from animal tissue.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] The present invention relates to a composition effective for cancer treatment.

Background Art

[0002] Plasmalogens are known to have effects such as promoting neurogenesis, suppressing neuroinflammation caused by lipopolysaccharide (LPS), and suppressing the accumulation of amyloid-β (Aβ) protein in the brain, and are said to be effective in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, depression, and schizophrenia. For example, Non-Patent Document 1 reports that the memory function of patients with mild Alzheimer's disease was improved by orally administering purified plasmalogen derived from scallops.

[0003] On the other hand, the number of cancer patients in Japan is increasing year by year, and it is said that one in two Japanese people will develop some form of cancer in their lifetime. There is also data indicating that one in three Japanese people die of cancer. Although the survival rate of cancer patients has increased due to advancements in medical technology and the like, more effective treatment means are still required.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[17] (2017) 199-205

Disclosure of the Invention

[0005] As described above, various reports have been made on plasmalogens, but the effects of plasmalogens on cancer have not been examined in detail.

[0006] An object of the present invention is to provide a composition or the like effective for cancer treatment. [Means for Solving the Problems]

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that plasmalogens can induce apoptosis of cancer cells and suppress the growth of cancer cells, and have completed the present invention.

[0008] That is, the present invention is as follows. [1] A composition for inducing apoptosis of cancer cells, characterized by containing a plasmalogen. [2] A composition for activating macrophages, characterized by containing a plasmalogen. [3] A composition for activating GPR21, characterized by containing a plasmalogen. [4] A composition for destroying the extracellular matrix in cancer tissues, characterized by containing a plasmalogen. [5] A composition for suppressing cancer cell growth, characterized by containing a plasmalogen. [6] A composition for cancer treatment, characterized by containing a plasmalogen.

[0009] [7] The composition for inducing apoptosis of cancer cells according to [1] above, wherein the plasmalogen is a plasmalogen extracted from animal tissues. [8] The composition for activating macrophages according to [2] above, wherein the plasmalogen is a plasmalogen extracted from animal tissues. [9] The composition for activating GPR21 according to [3], characterized in that the plasmalogen is a plasmalogen extracted from animal tissues.

[10] The composition for extracellular matrix destruction in cancer tissues according to [4], characterized in that the plasmalogen is a plasmalogen extracted from animal tissues.

[11] The composition for suppressing cancer cell proliferation according to the above [5], characterized in that the plasmalogen is a plasmalogen extracted from animal tissues.

[12] The composition for cancer treatment according to the above [6], characterized in that the plasmalogen is a plasmalogen extracted from animal tissues.

Advantages of the Invention

[0010] The composition of the present invention can induce apoptosis of cancer cells and suppress the proliferation of cancer cells, and is effective for the treatment of cancer.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] The composition of the present invention is characterized by containing plasmalogen. The composition of the present invention can induce apoptosis of cancer cells and suppress the growth of cancer cells by activating NK cells and macrophages. As a result, it is possible to suppress the enlargement and shrink the cancer tissue (malignant tumor tissue). In addition, the composition of the present invention can activate G protein-coupled receptor 21 (GPR21), and this activation is considered to contribute to the suppression of cancer cell growth. Furthermore, the composition of the present invention can destroy the extracellular matrix found in invasive cancer and the like.

[0013] Therefore, the composition of the present invention can be used as a composition for inducing apoptosis of cancer cells, a composition for activating macrophages, a composition for activating GPR21, a composition for destroying extracellular matrix in cancer tissues, a composition for suppressing cancer cell growth, a composition for suppressing cancer metastasis, a composition for treating cancer, etc. Specifically, for example, it can be used for alleviating symptoms and treating cancer patients.

[0014] Plasmalogen is a kind of phospholipid having an antioxidant action and is one of the glycerophospholipids. It is a subclass peculiar to glycerophospholipids characterized by having a vinyl ether bond at the sn-1 position of the glycerol backbone and has been confirmed at high concentrations in the cell membranes of many mammalian tissues.

[0015] The plasmalogen used in the present invention is not particularly limited as long as it is generally classified as a plasmalogen. For example, choline-type plasmalogen, ethanolamine-type plasmalogen, inositol-type plasmalogen, and serine-type plasmalogen can be mentioned. Among these, choline-type plasmalogen and ethanolamine-type plasmalogen are preferred, and ethanolamine-type plasmalogen is particularly preferred.

[0016] The plasmalogen of the present invention can be extracted from animal tissues. The animal tissues are not particularly limited as long as they contain plasmalogen, and examples include aquatic animals such as shellfish, sea squirts, sea cucumbers, salmon, saury, and skipjack, and birds. Among these, shellfish, sea squirts, and birds are preferred, and shellfish are particularly preferred. As the part to be used, an edible part (edible portion) is preferred. These animal tissues may be cut pieces, but it is preferable to use pulverized materials because plasmalogen can be extracted more efficiently.

[0017] Examples of shellfish include edible bivalves such as scallops, oysters, and abalones, and whelks, and scallops are particularly preferred. Scallops are edible bivalves belonging to the family Pectinidae, and examples include those belonging to the genus Mizuhopecten and the genus Pecten. Specifically, the Japanese scallop (scientific name: Mizuhopecten yessoensis) collected in Japan, the European scallop (scientific name: Pecten maximus (Linnaeus)) collected in Europe, etc. can be mentioned. Examples of the edible part include adductor muscles and strings.

[0018] A sea squirt is an edible chordate belonging to the family Styelidae, and examples include those belonging to the genus Halocynthia and the genus Akaholocynthia. Specifically, the Halocynthia roretzi (scientific name) and the Akaholocynthia aurantium (scientific name) can be mentioned. Examples of the edible part include the body part (fascicular body).

[0019] The birds are not particularly limited as long as they are edible birds, and examples include chickens, silky fowls, ducks, etc. As the edible part, breast meat rich in plasmalogen is preferred.

[0020] The extraction of plasmalogen can be carried out using water, an organic solvent, or a water-containing organic solvent, and it is preferable to use enzymatic treatment in combination. For example, an ethanol extraction method or a hexane extraction method can be mentioned, and the ethanol extraction method is preferred.

[0021] The ethanol extraction method is not particularly limited as long as it is a method of extracting using ethanol (including water-containing ethanol). For example, the methods described in JP-A-2019-140919, JP-A-2018-130130, Reprint 2012-039472, JP-A-2010-065167, JP-A-2010-063406, etc. can be mentioned.

[0022] The hexane extraction method is not particularly limited as long as it is a method of extracting using hexane. For example, the methods described in Reprint 2009-154309, Reprint 2008-146942, etc. can be mentioned.

[0023] The composition of the present invention can be used as a pharmaceutical. It can also be used as a food for assisting cancer treatment (including so-called health foods such as foods for specified health use, foods with nutritional functions, and foods with functional claims).

[0024] The composition of the present invention may be for oral use or parenteral use such as injection or infusion. However, from the viewpoint of easy intake, oral use is preferred. In the case of oral use, examples of its form include tablet form, capsule form, powder form, granule form, liquid form, granule form, bar form, plate form, block form, solid form, round form, paste form, cream form, caplet form, gel form, chewable form, stick form, etc. Among these, the capsule form is preferred.

[0025] As for the content of plasmalogen in the composition of the present invention, it may be appropriately contained within the range where the effect is exhibited. Although it depends on the form, for example, when converted to dry mass, plasmalogen is 10 -10 mass% or more of the whole composition of the present invention, preferably 10 -5 mass% or more, more preferably 0.1 mass% or more, and particularly preferably 1.0 mass% or more.

[0026] There is no particular limitation on the intake amount of the oral composition of the present invention. From the viewpoint of more significantly exhibiting the effect of the present invention, the intake amount of plasmalogen is preferably 10 -6 μg / day or more per adult per day, more preferably 1 μg / day or more, further preferably 500 μg / day or more, and particularly preferably 1000 μg / day or more. The upper limit is, for example, 20,000 μg / day, preferably 10,000 μg / day.

[0027] The oral composition of the present invention can be contained as one-day supply in one container or divided into, for example, 2 to 3 plural containers so that the intake amount per day becomes the above intake amount.

[0028] The composition of the present invention can be produced by a known method by adding other components other than the components of the present invention, if necessary. Examples of other components other than the components of the present invention include vitamins, minerals, proteins, peptides, amino acids, animal oils, and vegetable oils.

[0029] Hereinafter, the present invention will be described in detail based on examples.

Examples

[0030] The tumor suppression effect of mice by plasmalogen administration was examined.

[0031] [Plasmalogen] Plasmalogen (sPls) was prepared from scallops (scientific name: Mizuhopecten yessoensis) using a hexane-extracted ethanolamine-type plasmalogen (mainly containing ethanolamine-type plasmalogen and also containing choline-type plasmalogen) prepared by the following method.

[0032] 1. Add Kokrase P (manufactured by Mitsubishi Chemical Foods Corporation) and phospholipase A1 (PLA1) (manufactured by Mitsubishi Chemical Foods Corporation) to fresh scallop strings and mix well. 2. Next, add hexane / isopropanol and aspirate and filter the supernatant. 3. Add an aqueous sodium sulfate solution and mix well. 4. Dry the upper layer with a rotary evaporator until dry. 5. Add acetone cooled to 4°C and mix well. 6. Centrifuge at 3000 rpm, 10 min, 4°C. 7. Discard the supernatant and collect the precipitate. 8. Dry overnight in a desiccator.

[0033] [In vivo xenograft tumor transplantation experiment] An in vivo xenograft tumor transplantation experiment was performed using SCID (Severe Combined Immunodeficiency) mice that lack functional T cells and B cells in peripheral blood and exhibit severe combined immunodeficiency.

[0034] Eight-week-old SCID mice were orally administered sPls suspended in ultrapure water at a dose of 0.02 mg / kg / day (in terms of sPls) for 6 weeks. The control group was orally administered ultrapure water for 6 weeks. Both the sPls administration group and the control group had 5 mice / group and were housed in a SPF (Specific pathogen free) environment throughout the entire experimental period.

[0035] [Transplantation of cancer cells] Cancer cells (human neuroblastoma SH-SY5Y cells) cultured in DEME medium containing 10% FBS, 5×10 6100 μL of the cell suspension at cells / mL was mixed with Matrigel (basement membrane matrix, collagen type 1) and subcutaneously transplanted onto the back of each mouse that had been orally administered the above sPls or ultrapure water for 6 weeks. Four weeks after transplantation of SH-SY5Y cells, tumors were excised from each mouse and weighed and sized.

[0036] Figure 1A shows the sizes of the tumors excised from each mouse in the control group and the sPls-administered group. Figure 1B shows the average value of the weights (mg) of the excised tumors and the results of the comparative test by Student's t-test.

[0037] As shown in Figure 1A, a significant reduction in tumor size was observed in the sPls-administered group compared to the control group. Also, as shown in Figure 1B, a statistically significant decrease in tumor weight was observed in the sPls-administered group compared to the control group. Therefore, it became clear that the administration of plasmalogen suppresses the growth of tumor cells.

Example

[0038] Tissue specimens of the tumors of each mouse excised in Example 1 were prepared to examine the induction of apoptosis of tumor cells by plasmalogen. Specifically, the following operations were performed.

[0039] 1. Wash the tumor tissues excised from each mouse in the sPls-administered group and the control group with phosphate buffered saline (PBS). 2. Fix with 4% paraformaldehyde. 3. Embed the fixed tumor tissues in an embedding agent for cryosectioning and freeze at -80°C. 4. Prepare tissue specimens with a thickness of 10 μm using a Cryostat Microm HM550. 5. Using an In Situ Cell Death Detection Kit (Roche Diagnostics) by the TUNEL method, label the fragmented DNA due to apoptosis in the tissue specimens with biotin-labeled nucleotides, and then react with TUNEL and HRP-labeled streptavidin for staining. 6. Stain the cell nuclei with DAPI.

[0040] Figure 2A shows the localization of apoptotic cells in the tumor tissues of mice in the control group and the sPls administration group. Figure 2B shows the results of a comparative test by Student's t-test for the number of TUNEL-positive cells in the control group and the sPls administration group.

[0041] As shown in Figure 2A, in the sPls administration group, more TUNEL-positive cells were observed in the tumor tissue compared to the control group. Also, as shown in Figure 2B, in the sPls administration group, it was confirmed that the number of TUNEL-positive cells was statistically significantly increased compared to the control group. Therefore, it was suggested that plasmalogen induces apoptosis of cancer cells and has a tumor-reducing effect.

Example

[0042] Cancer cells cause an increase in type I collagen production, cross-linking proliferation of collagen fibers, and an increase in cellular fibronectin from fibroblasts in the tumor stroma, and harden and alter the extracellular matrix around cancer cells. On the other hand, the extracellular matrix with enhanced hardness acts on cancer cells as mechanical stress and is involved in enhancing the proliferation, invasion, and metastasis ability of cancer. Thus, since the interaction between cancer cells and the extracellular matrix is deeply related to the progression of tumors, the changes in the extracellular matrix by plasmalogen were examined for the tumor tissues of each mouse in the sPls administration group and the control group. Specifically, the following operations were performed.

[0043] 1. Stain the cell nuclei of the tumor tissue specimens with hematoxylin. 2. After washing with water, stain the cytoplasm with eosin. 3. Dehydrate with an ethanol series and clear with xylene. 4. Drop a mounting agent and seal with a coverslip to prepare hematoxylin and eosin staining specimens. 5. Perform histological observation of the hematoxylin and eosin staining specimens with an optical microscope.

[0044] Figure 3A shows the extracellular matrix in tumor tissues stained with hematoxylin and eosin for mice in the control group and the sPls-administered group. Figure 3B quantifies the extracellular matrix fragmented from tumor sites of the same area and shows the results of a comparative test by Student's t-test.

[0045] As shown in Figure 3A, in the tumor tissues of mice in the control group, the extracellular matrix commonly seen in invasive tumors was clearly observed. However, in the tumor tissues of mice in the sPls-administered group, fragmentation of the extracellular matrix seen in the regression phase of the tumor was confirmed. Also, as shown in Figure 3B, it was confirmed that the relative number of fragmented extracellular matrix was statistically significantly increased. Therefore, it was suggested that plasmalogen is effective for cancer metastasis and cancer treatment.

Example

[0046] The expression of the plasmalogen receptor, G protein-coupled receptor 21 (GPR21), was examined in the tumor tissues of each mouse in the sPls-administered group and the control group. Specifically, the following operations were performed.

[0047] Using a GPR21 antibody (Invitrogen) as the primary antibody and FITC-conjugated anti-rabbit IgG as the secondary antibody, the detection of GPR21-positive cells in mouse tumor tissues was performed by immunohistochemical methods. GPR21-positive cells were observed with a fluorescence microscope (Axopskope 2, Zeiss).

[0048] Figure 4A shows the localization of GPR21-positive cells in the tumor tissues of mice in the control group and the sPls-administered group. Figure 4B shows the results of a comparative test by Student's t-test of the relative number of GPR21-positive cells in the control group and the sPls-administered group.

[0049] As shown in Figure 4B, in the sPls-administered group, it was confirmed that the expression level of GPR21 was statistically significantly increased in the tumor tissues compared to the control group.

Example

[0050] Peripheral blood lymphocytes are composed of three cell populations: T cells, B cells, and natural killer cells (NK cells), and play a central role in immune function. The expression of CD16 antigen is related to the cytotoxic activity of NK cells. NK cells showing CD16 positivity exhibit strong cytotoxic activity and play an essential role in the destruction of malignant tumor cells, etc. Regarding the tumor tissues of each mouse in the sPls administration group and the control group, the increase in CD16-positive NK cells in the tumor tissue by plasmalogen was examined. Specifically, the following operations were performed.

[0051] Using rabbit GPR21 antibody (Invitrogen) and rat CD16 antibody (BD Pharmingen), CD16-positive NK cells in mouse tumor tissues were detected by immunohistochemical methods. CD16-positive NK cells were observed with a fluorescence microscope (Axioskope 2, Zeiss).

[0052] Figure 5A shows the results of immunohistochemical staining of CD16-positive activated NK cells, GPR21-positive cells, and the combination of both in the tumor tissues of the control group and the sPls administration group. Figure 5B shows the results of a comparative test by Student's t-test of the relative numbers of CD16-positive cells in the control group and the sPls administration group.

[0053] As shown in Figure 5A, an increase in CD16-positive NK cells was confirmed in the tumor tissues of the sPls administration group. Also, it was observed that GPR21, the sPls receptor, was highly expressed in NK cells. As shown in Figure 5B, in the sPls administration group, it was confirmed that CD16-positive NK cells were statistically significantly increased in the tumor tissues compared to the control group.

Example

[0054] Regarding the tumor tissues of each mouse in the sPls administration group and the control group, the increase in F4 / 80-positive activated macrophages in the tumor tissues by plasmalogen was examined. Specifically, the following operations were performed.

[0055] Using an anti-F4 / 80 antibody (abcam, ab16911), which is a major marker of mature macrophages in mice, the detection of F4 / 80-positive activated macrophages in the tumor tissues of mice was performed by immunohistochemical methods. F4 / 80-positive activated macrophages were observed with a fluorescence microscope (Axioskope 2, Zeiss).

[0056] Figure 6A shows the results of immunohistochemical staining of F4 / 80-positive activated macrophages, GPR21-positive cells, and the combination of both in the tumor tissues of each mouse in the control group and the sPls administration group. Figure 6B shows the results of a comparative test by Student's t-test of the relative numbers of F4 / 80-positive cells in the control group and the sPls administration group.

[0057] As shown in Figure 6A, in the tumor tissues of the sPls administration group, a significant increase in F4 / 80-positive activated macrophages was observed, and they were found to accumulate in tumor cells. Furthermore, co-staining of F4 / 80-positive activated macrophages and GPR21-positive cells suggested that GPR21 was highly expressed in activated macrophages. Also, as shown in Figure 6B, quantification of F4 / 80-positive activated macrophages showed that the number of activated macrophages in the sPls administration group was statistically significantly increased compared to the control group.

[0058] [Formulation Example] Hard capsules were manufactured according to the following formulation. Scallop-extracted plasmalogen 0.5 mg Cyclodextrin 3.3 mg Amino acid 1.2 mg Pine dextrin 185.0 mg [Industrial Applicability]

[0059] Since the composition of the present invention is effective for cancer treatment, it is industrially useful.

Claims

1. A composition for inducing apoptosis of cancer cells, characterized by containing plasmalogen.

2. A composition for activating macrophages, characterized by containing plasmalogen.

3. A composition for activating GPR21, characterized by containing plasmalogen.

4. A composition for destroying extracellular matrix in cancer tissues, characterized by containing plasmalogen.

5. A composition for inhibiting cancer cell proliferation, characterized by containing plasmalogen.

6. A composition for treating cancer, characterized by containing plasmalogen.

7. The composition for inducing apoptosis of cancer cells according to Claim 1, wherein the plasmalogen is a plasmalogen extracted from animal tissues.

8. The composition for activating macrophages according to Claim 2, wherein the plasmalogen is a plasmalogen extracted from animal tissues.

9. The composition for activating GPR21 according to Claim 3, wherein the plasmalogen is a plasmalogen extracted from animal tissues.

10. The composition for destroying extracellular matrix in cancer tissues according to Claim 4, wherein the plasmalogen is a plasmalogen extracted from animal tissues.

11. The composition for inhibiting cancer cell proliferation according to Claim 5, wherein the plasmalogen is a plasmalogen extracted from animal tissues.

12. The composition for treating cancer according to Claim 6, wherein the plasmalogen is a plasmalogen extracted from animal tissues.

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