Immuno-anticancer composition comprising toxicodendron vernicifluum extract and cytokine-induced killer cells

Abstract

It has been conventionally known that a Toxicodendron vernicifluum extract is effective in treating arthritis and the like by suppressing immunity. However, in the present invention, it has been experimentally demonstrated that treatment of cytokine-induced killer cells with a Toxicodendron vernicifluum extract actually increases the immune sensitivity of immune cells, resulting in improved cancer cell killing ability. Therefore, an immune-anticancer composition comprising a Toxicodendron vernicifluum extract and cytokine-induced killer cells as active ingredients, of the present invention, can effectively treat lung cancer or breast cancer by means of improved cancer cell killing ability, and, when prepared, an anticancer immune cell therapeutic preparation comprising same can be administered to patients with lung cancer and patients with breast cancer so as to effectively treat lung cancer and breast cancer.

Description

An immuno-oncology composition comprising lacquer tree extract and cytokine-induced killer cells

[0001] The present invention provides an immuno-cancer composition comprising a lacquer tree extract and cytokine-induced killer cells as active ingredients, and an anti-cancer immune cell therapy composition comprising the same.

[0002] The lacquer tree (Toxicodendron Vernicifluum or Rhus Veniciflua Strokes) is a deciduous broadleaf small tree belonging to the Anacardiaceae family, and is known to be native to the Central Asian highlands and the Himalayas.

[0003] The sap of the lacquer tree is called lacquer, and in oriental medicine, it has been known since ancient times that dried lacquer removes blood stasis, promotes blood circulation, and is effective for treating parasites, abdominal pain, hyperacidity, pulmonary tuberculosis, menstrual cramps, constipation, diabetes, dysentery, antiseptic, strengthens the stomach, and relieves female meridian blockage.

[0004] Poison ivy contains a large amount of antioxidants. According to known results, antioxidants found in poison ivy bark extract have been shown to exhibit strong antioxidant activity in rat brain cells and inhibit the growth of blood cancer cells.

[0005] In addition, Korean Patent Application No. 10-1997-0013163, Korean Patent Application No. 10-1997-0004193, and Korean Patent Registration No. 10-0251526 report that the urushiol component of lacquer extract has an anticancer effect; and Korean Patent Registration No. 10-0257448 reports an anticancer composition comprising fustin, fisetin, sulfuretin, and butein as active ingredients in a composition purified from lacquer tree extract using a silica column.

[0006] Meanwhile, Korean Patent No. 10-0807169 reported the use of a lacquer tree extract as a composition for the prevention and treatment of arthritis. The patent document reported that lacquer tree extract exhibits pharmacological effects, including immunosuppressive effects, synovial cell activation inhibition, anti-inflammatory effects, and preventative and therapeutic effects in animal models of arthritis.

[0007] The above results show that the lacquer tree extract has an anticancer effect when administered to a cancer animal model or directly treated with a cell line, and that the lacquer tree extract has an anti-inflammatory effect and an effect in treating arthritis by suppressing the immune response.

[0008] Therefore, it has been determined that lacquer tree extract induces anticancer effects through mechanisms other than the immune response, such as directly inducing cancer cell death or activating the immune response.

[0009] The patent documents and references mentioned in this specification are incorporated herein by reference to the same extent as if each document were individually and specifically identified by reference.

[0010] The present invention experimentally demonstrates that treating cytokine-induced killer cells with a lacquer tree extract enhances their cancer-killing capacity. Accordingly, the present invention provides an immuno-cancer composition comprising a lacquer tree extract and cytokine-induced killer cells as active ingredients, and an anticancer immune cell therapy composition comprising the same.

[0011] Other objects and technical features of the present invention are presented more specifically in the detailed description of the invention, the claims and the drawings below.

[0012] The present invention provides an immuno-anticancer composition comprising a lacquer tree extract and cytokine-induced killer cells as active ingredients.

[0013] The above lacquer tree extract is characterized by enhancing the killing ability of cytokine-induced killer cells against cancer cells, and the cancer cells are characterized by being lung cancer or breast cancer cells.

[0014] When the cancer cells (cells / ml) are treated with 5 to 10 times the cytokine-induced killer cells (cells / ml) and simultaneously treated with the lacquer tree extract at 10 to 100 ㎍ / ml, the activity of the cancer cells is further reduced by 5 to 30% compared to when the lacquer tree extract is not treated.

[0015] The cancer cells are characterized as lung cancer or breast cancer cells; and the cytokine-induced killer cells are 5x10 4 5x10 5 cells / ㎖ and 10㎍ / ㎖ of lacquer tree extract were administered to 5x10 lung cancer cells 4 When treated simultaneously at 10 cells / ㎖, the activity of the cancer cells is further reduced by 20 to 30% compared to when the lacquer tree extract is not treated; 5x10 of the cytokine-induced killer cells 4 2.5x10 5 cells / ㎖ and 10 to 100 ㎍ / ㎖ of lacquer tree extract were added to 5x10 breast cancer cells. 4 When treated simultaneously at 100 cells / ㎖, the activity of the cancer cells is further reduced by 5 to 20% compared to when the lacquer tree extract is not treated.

[0016] Previously, it was known that lacquer tree extracts were effective in treating conditions such as arthritis by suppressing the immune system. However, the present invention experimentally demonstrated that treating cytokine-induced killer cells with lacquer tree extracts actually increased the immune cell sensitivity, resulting in enhanced cancer cell killing capacity.

[0017] Therefore, the immuno-anticancer composition comprising the lacquer tree extract of the present invention and cytokine-induced killer cells as active ingredients has the advantage of being able to effectively treat lung cancer or breast cancer due to enhanced cancer cell killing ability, and when an anticancer immune cell therapy agent comprising the same is manufactured, it can be administered to lung cancer and breast cancer patients to effectively treat lung cancer and breast cancer.

[0018] Figure 1 shows changes in the killing effect of cytokine-induced killer cells treated with the lacquer tree extract (RVS extract) of the present invention on lung cancer cell lines (H441).

[0019] Figure 2 shows changes in the apoptotic effect of cytokine-induced killer cells treated with the RVS extract of the present invention on breast cancer cell lines (MCF7).

[0020] Figure 3 shows changes in the killing effect of cytokine-induced killer cells treated with the lacquer tree extract of the present invention on breast cancer cell lines (MDA-MB231).

[0021] The present invention provides an immuno-anticancer composition comprising a lacquer tree extract and cytokine-induced killer cells as active ingredients.

[0022] The above lacquer tree extract can be prepared by a method of hot water extraction using a solvent selected from water, methanol, ethanol, butanol, or a mixed solvent thereof, such as the bark of the lacquer tree. Specifically, the bark of the lacquer tree is cut into pieces, dried, and then heat-extracted in water at 95°C for 2 hours, cooled, and the supernatant is filtered through a filter with a diameter of 0.2 μm, concentrated under reduced pressure, and then freeze-dried to obtain the extract.

[0023] The above cytokine-induced killer cells are a type of immune cell that recognizes infected cells or cancer cells, attaches to them, and secretes enzymes that destroy the outer membrane of the cells, causing them to die.

[0024] The lacquer tree extract of the present invention is characterized by enhancing the killing ability of cytokine-induced killer cells against cancer cells, and the cancer cells are not limited to any cancer cells that are monitored and killed by cytokine-induced killer cells, but are preferably lung cancer or breast cancer cells.

[0025] According to an embodiment of the present invention, the present invention is characterized in that when the cancer cells (cells / ml) are treated with 5 to 10 times the cytokine-induced killer cells (cells / ml) and simultaneously treated with the lacquer tree extract at 10 to 100 ㎍ / ml, the activity of the cancer cells is further reduced by 5 to 30% compared to when the lacquer tree extract is not treated.

[0026] In detail, 5x10 of the cytokine-induced killer cells 4 5x10 5 cells / ㎖ and 10㎍ / ㎖ of lacquer tree extract were administered to 5x10 lung cancer cells 4 When treated simultaneously at 100 cells / ㎖, the activity of the cancer cells is further reduced by 20 to 30% compared to when the lacquer tree extract is not treated; 5x10 of the cytokine-induced killer cells 4 2.5x10 5 cells / ㎖ and 10 to 100 ㎍ / ㎖ of lacquer tree extract were added to 5x10 breast cancer cells. 4 When treated simultaneously at 100 cells / ㎖, the activity of the cancer cells is further reduced by 5 to 20% compared to when the lacquer tree extract is not treated.

[0027] The immuno-anticancer composition of the present invention can produce 5x10 cytokine-induced killer cells 4 5x10 5 It can be prepared at a ratio of 10 to 100 μg / ml of cells / ml and lacquer extract, and if the constant ratio is maintained, there is no problem even if an appropriate carrier is used in addition to the buffer solution to maintain the activity of the cells.

[0028] The immuno-anticancer composition of the present invention includes cytokine-induced killer cells that kill cancer cells and a lacquer tree extract that enhances the cancer cell killing ability of the cytokine-induced killer cells, and can be manufactured into an anticancer immune cell therapy agent including the immuno-anticancer composition.

[0029] The anticancer immune cell therapy of the present invention comprises cytokine-induced killer cells activated by a lacquer tree extract. The cytokine-induced killer cells can be prepared by isolating peripheral blood mononuclear cells (PBMCs) from peripheral blood, isolating cytokine-induced killer cells from the isolated PBMCs, and then treating the PBMCs with a lacquer tree extract to activate them. The activation is performed by treating the isolated cytokine-induced killer cells with a lacquer tree extract, and 5x10 of the cytokine-induced killer cells are prepared. 4 5x10 5 It is preferable to treat by culturing by administering 10 to 100 μg / ml of lacquer tree extract to cells / ml.

[0030] The immuno-oncology composition of the present invention can be prepared as an anticancer immune cell therapy composition. The anticancer immune cell therapy composition of the present invention is not limited to any cancer known to be treatable by cytokine-induced killer cells, but lung cancer or breast cancer is preferred.

[0031] The anticancer immune cell therapy composition of the present invention can be manufactured in a unit dosage form by formulating it using a pharmaceutically acceptable excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains. At this time, the formulation may be in the form of a suspension in a cell freezing solution or a buffer solution, and may additionally include a stabilizer. The anticancer immune cell therapy composition of the present invention can be administered parenterally, and can also be administered by intravenous injection, subcutaneous injection, intraperitoneal injection, transdermal administration, etc. The appropriate dosage of the anticancer immune cell therapy composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, administration time, and administration route.

[0032]

[0033] The present invention is described in detail below through examples.

[0034]

[0035] Example

[0036]

[0037] 1. Cytokine-induced killer cell-mediated apoptosis of lung cancer cell lines by lacquer tree extract

[0038] The change in cytokine-induced killer cell (CIK) apoptosis effect of lacquer tree (Toxicodendron Vernicifluum or Rhus Veniciflua Strokes) extract on lung cancer cell line (H441) was confirmed.

[0039] The above lacquer tree extract (RVS extract) was prepared by hot water extraction, filtration, concentration under reduced pressure, and freeze-drying; the CIK was prepared by culturing human PBMC with CD3 antibody and IL-2 in T-cell culture medium for 2 weeks; the lung cancer cell line (H441) was cultured by adding 10% FBS to RPMI 1604 medium, and 5x10 were seeded in a 96-well plate one day before the CIK co-culture. 4 Seeding was done at a concentration of 10 cells / ㎖.

[0040] Lung cancer cell activity was analyzed using the RFP killing assay. The RFP killing assay is a method for measuring cell killing ability through immune cells by measuring the decrease in RFP-positive cells due to the killing effect when co-cultured with CIK after generating RFP stable cell lines using H441, MCF7, and MDA-MB-231.

[0041] Figure 1 shows changes in the killing effect of cytokine-induced killer cells treated with the lacquer extract of the present invention on lung cancer cell lines (H441).

[0042] Lung cancer cell line (H441, 5x10 4 cells / ㎖) treated with 10 ㎍ / ㎖ of RVS extract and cultured for 48 hours, and lung cancer cell line (H441, 5x10 cells / ㎖) cultured for 48 hours without treatment with RVS extract 4 As a result of comparing the cell activity (cell / ㎖), it was confirmed that the cell activity was the same (see ns in Fig. 1). Therefore, it is determined that simply treating the RVS extract of the present invention to the lung cancer cell line (H441) does not change the activity of the lung cancer cell line (H441).

[0043] Lung cancer cell line (H441, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 4 cells / ㎖) and cultured for 48 hours, the lung cancer cell line (H441, 5x104 It was confirmed that the lung cancer cell line activity (H441, RFP count) decreased by approximately 13% compared to the results of culturing only (10 cells / ㎖) for 48 hours.

[0044] Lung cancer cell line (H441, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 4 cells / ㎖) and RVS 10㎍ / ㎖ were simultaneously treated and cultured for 48 hours. As a result, lung cancer cell line (H441, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 4 It was confirmed that the lung cancer cell line activity (H441, RFP count) was reduced by approximately 21% more than the results obtained by treating only (10 cells / ㎖) and culturing for 48 hours (see ** in Fig. 1).

[0045] Also, lung cancer cell lines (H441, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 5 cells / ㎖) and RVS extract 10 ㎍ / ㎖ were simultaneously treated and cultured for 48 hours, and the lung cancer cell line (H441, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 5 It was confirmed that the lung cancer cell line activity (H441, RFP count) was reduced by approximately 25% more than the results obtained when only lung cancer cell line (lung cancer cell line:cytokine-induced killer cells = 1:10) was treated and cultured for 48 hours (see * in Figure 1).

[0046] Therefore, it is believed that the RVS extract of the present invention has no direct cell killing ability against lung cancer cell line (H441), but when used together with cytokine-induced killer cells, it enhances the killing ability of lung cancer cell line (H441) by cytokine-induced killer cells.

[0047]

[0048] 2. Cytokine-induced killer cell-mediated apoptosis of breast cancer cell lines by lacquer tree extract

[0049] The change in the cytokine-induced killer cell (CIK) killing effect of the RVS extract of the present invention on breast cancer cell line (MCF7) was confirmed.

[0050] Figure 2 shows changes in the apoptotic effect of cytokine-induced killer cells treated with the RVS extract of the present invention on breast cancer cell lines (MCF7).

[0051] Breast cancer cell line (MCF7, 5x10 4 cells / ㎖) treated with 10 ㎍ / ㎖ of RVS extract and cultured for 48 hours, and breast cancer cell line (MCF7, 5x10 cells / ㎖) cultured for 48 hours without treatment with RVS extract 4 As a result of comparing the cell activity (cells / ㎖), it was confirmed that the cell activity was the same.

[0052] Therefore, it is judged that simply treating the RVS extract of the present invention to the breast cancer cell line (MCF7) does not change the activity of the breast cancer cells (MCF7).

[0053] Breast cancer cell line (MCF7, 5x10 4 cells / ㎖) in cytokine-induced killer cells (2.5x10 5 cells / ㎖) and cultured for 48 hours, the breast cancer cell line (MCF7, 5x10 4 Compared to the results of culturing only (10 cells / ㎖) for 48 hours, it was confirmed that the activity of breast cancer cell lines (MCF7, RFP count) was reduced by approximately 62%.

[0054] Breast cancer cell line (MCF7, 5x10 4 cells / ㎖) in cytokine-induced killer cells (2.5x10 5 cells / ㎖) and RVS extract 10 ㎍ / ㎖ were simultaneously treated and cultured for 48 hours, and the breast cancer cell line (MCF7, 5x10 4 cells / ㎖) in cytokine-induced killer cells (2.5x10 5It was confirmed that the activity of breast cancer cell lines (MCF7, RFP count) was reduced by approximately 5% more than the results obtained after treating (10 cells / ㎖) and culturing for 48 hours (see * in Fig. 2).

[0055] Figure 3 shows changes in the killing effect of cytokine-induced killer cells treated with the lacquer extract of the present invention on breast cancer cell lines (MDA-MB231).

[0056] The breast cancer cell line (MDA-MB231) differs from the breast cancer cell line (MCF7) in origin and receptor expression level. The breast cancer cell line (MDA-MB231) is derived from adenocarcinoma and is characterized by not expressing estrogen receptor, progesterone receptor, or HER2 (CD340) and by having a confirmed P53 mutation. In contrast, the breast cancer cell line (MCF7) is derived from invasive ductal carcinoma and is characterized by expressing estrogen receptor and progesterone receptor, but not HER2 (CD340) and by having no confirmed P53 mutation.

[0057] Experimental results showed that breast cancer cell lines (MDA-MB231, 5x10 4 cells / ㎖) treated with 100 ㎍ / ㎖ of RVS extract and cultured for 48 hours, and breast cancer cell line (MDA-MB231, 5x10 cells / ㎖) cultured for 48 hours without treatment with RVS extract 4 As a result of comparing the cell activity (cells / ㎖), it was confirmed that the cell activity was the same.

[0058] Breast cancer cell line (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 4cells / ㎖) and RVS extract 0 ㎍ / ㎖, RVS extract 10 ㎍ / ㎖, or RVS extract 100 ㎍ / ㎖ were treated simultaneously and cultured for 48 hours to analyze cell activity.

[0059] Experimental results showed that breast cancer cell lines (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 4 When only 5x10 cells / ㎖) were treated, breast cancer cell lines (MDA-MB231, 5x10 4 cells / ㎖) for 48 hours or breast cancer cell line (MDA-MB231, 5x10 4 It was confirmed that the activity of breast cancer cell lines (MDA-MB231, RFP count) decreased by approximately 35% compared to the results of culturing them by treating them with 100 μg / ml of RVS extract and 100 μg / ml of RVS extract.

[0060] Breast cancer cell line (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 4 cells / ㎖) and RVS 10㎍ / ㎖ were simultaneously treated and cultured for 48 hours. As a result, the breast cancer cell line (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 4 It was confirmed that the activity of breast cancer cell line (MDA-MB231, RFP count) was reduced by about 5% more than the result of treating only 10 cells / ㎖ and culturing for 48 hours; breast cancer cell line (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 4 cells / ㎖) and RVS extract 100 ㎍ / ㎖ were simultaneously treated and cultured for 48 hours, and the breast cancer cell line (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (5x10 4It was confirmed that the activity of breast cancer cell lines (MDA-MB231, RFP count) was reduced by approximately 15% compared to the results obtained when only (cells / ㎖) was treated and cultured for 48 hours.

[0061] Breast cancer cell line (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (2.5x10 5 Cell activity was analyzed by treating 0 μg / ml of RVS extract, 10 μg / ml of RVS extract, or 100 μg / ml of RVS extract and culturing for 48 hours.

[0062] Experimental results showed that breast cancer cell lines (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (2.5x10 5 When only 5x10 cells / ㎖) were treated, breast cancer cell lines (MDA-MB231, 5x10 4 It was confirmed that the activity of breast cancer cell line (MDA-MB231, RFP count) was reduced by approximately 62% compared to the results of culturing only (10 cells / ㎖) for 48 hours.

[0063] Breast cancer cell line (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (2.5x10 5 cells / ㎖) and RVS extract 10 ㎍ / ㎖ were simultaneously treated and cultured for 48 hours. As a result, breast cancer cell line (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (2.5x10 5 It was confirmed that the activity of breast cancer cell line (MDA-MB231, RFP count) was reduced by approximately 13% more than the result of treating only 5 cells / ㎖ and culturing for 48 hours; breast cancer cell line (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (2.5x10 5cells / ㎖) and RVS extract 100 ㎍ / ㎖ were simultaneously treated and cultured for 48 hours, and the breast cancer cell line (MDA-MB231, 5x10 4 cells / ㎖) in cytokine-induced killer cells (2.5x10 5 It was confirmed that the activity of breast cancer cell lines (MDA-MB231, RFP count) was reduced by approximately 17% compared to the results obtained when only (cells / ㎖) was treated and cultured for 48 hours.

[0064] Therefore, the RVS extract of the present invention does not have direct apoptotic ability against breast cancer cell lines (MCF7 or MDA-MB231), but when used together with cytokine-induced killer cells, it is judged to enhance the apoptotic ability of breast cancer cell lines (MCF7 or MDA-MB231) by cytokine-induced killer cells, and it is judged that the apoptotic ability of breast cancer cell lines (MCF7 or MDA-MB231) by cytokine-induced killer cells is enhanced depending on the concentration of the RVS extract.

[0065] The specific embodiments described herein are intended to represent preferred embodiments or examples of the present invention and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that variations and other uses of the present invention do not depart from the scope of the invention described in the claims of this specification.

[0066]

[0067] The immuno-anticancer composition comprising the lacquer tree extract of the present invention and cytokine-induced killer cells as active ingredients has the advantage of being able to effectively treat lung cancer or breast cancer due to enhanced cancer cell killing ability, and when an anticancer immune cell therapy agent comprising the same is manufactured, it can be administered to lung cancer and breast cancer patients to effectively treat lung cancer and breast cancer.

Claims

1. An immuno-anticancer composition comprising lacquer tree extract and cytokine-induced killer cells as active ingredients, The above lacquer tree extract is an immuno-anticancer composition characterized by enhancing the killing ability of cytokine-induced killer cells against lung cancer cells and the killing ability against breast cancer cells. 5x10 of the above cytokine-induced killer cells 4 2.5x10 5 cells / ㎖ and 10 to 100 ㎍ / ㎖ of lacquer tree extract were added to 5x10 breast cancer cells. 4 An immuno-anticancer composition characterized in that, when treated simultaneously at a dose of 100 cells / ㎖, the activity of breast cancer cells is further reduced by 5 to 20% compared to when the lacquer tree extract is not treated.

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