Eutrema japonica leaf alcohol extract and use thereof in treatment of cancer stem cell-related cancer

Abstract

Disclosed is use of a pharmaceutical composition in the manufacture of a medicament for treating a cancer stem cell-related cancer, inhibiting the recurrence or metastasis of cancer stem cells after cancer tumor resection, or inhibiting tumor-associated angiogenesis. The pharmaceutical composition comprises an Eutrema japonica leaf alcohol extract. The cancer stem cell-related cancer comprises triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

Description

Wasabi leaf alcohol extract and its use in the treatment of cancer stem cell-related cancers. Technical Field

[0001] This invention relates to a pharmaceutical composition and its uses, and particularly to a pharmaceutical composition comprising an alcoholic extract of wasabi (Eutremajaponica) leaves, which can be used to treat cancer stem cell-related cancers, as adjuvant therapy after surgery, and to inhibit angiogenesis. Background Technology

[0002] Breast cancer is the leading cause of cancer incidence among women in Taiwan, with triple-negative breast cancer (TNBC) being the most challenging to treat. Ovarian cancer ranks fifth in cancer mortality among women in Taiwan. Both of these cancers have extremely poor overall prognoses and high recurrence rates, and currently there are no effective targeted therapies, making them the leading cause of death among gynecological cancers. In Taiwanese men, prostate cancer has seen a rising trend in both incidence and mortality, currently ranking sixth among the top ten causes of cancer death. Non-small cell lung cancer accounts for approximately 80-90% of all lung cancers. While recent advancements in medical technology have significantly improved survival rates, many cases still present challenges due to drug resistance. Oral cancer is the most common malignant tumor among head and neck cancers, with a global incidence of 3.8%. Although early detection leads to effective treatment, a recurrence rate as high as 30-50% still exists after surgery, adjuvant radiotherapy, or chemotherapy. Therefore, the above data highlight the crucial importance of developing novel and effective therapies for cancer.

[0003] Recent studies have pointed to a possible link between cancer recurrence and cancer stem cells (CSCs). These cells possess self-renewal and differentiation capabilities and exhibit specific cancer stem cell markers, such as ALDH1A1, CD44, Nanog, and EpCAM. Although CSCs constitute only a very small percentage of tumors, they are highly resistant to chemotherapy and radiotherapy unless all are eliminated, thus surviving and causing cancer recurrence. Cancer stem cells play a central role in tumorigenesis. The growth and differentiation of normal stem cells are regulated by related genes. Exposure to harmful substances, such as ultraviolet radiation and chemical substances, can potentially transform normal stem cells into cancer stem cells. The presence of cancer stem cells (CSCs) within tumors is considered a crucial and challenging issue in cancer treatment. Research has demonstrated that several signaling pathways, the tumor microenvironment, and mRNA can participate in regulating cancer stem cells, including enhancing self-renewal capacity (Notch, Wnt, and Hh signaling pathways), enhancing anti-apoptotic capacity (PI3K signaling pathway), enhancing antioxidant capacity (NRF2 signaling pathway), increasing anti-cancer drug activity (ABCG2), resisting hypoxia (HIF-1 signaling), and maintaining a tumor-friendly microenvironment (exosomes or chemokines). Breast cancer stem cells (BCSCs) play a key role in the development, metastasis, and drug resistance of breast cancer, thus necessitating the development of novel therapies that inhibit cancer stem cells.

[0004] Wasabi (Wasabia japonica or Eutremajaponicum) is a spicy spice commonly used in Japanese food. The whole plant is edible, but the leaves, which are usually discarded, actually have excellent antioxidant and antibacterial activities. 5-Hydroxyferulic acid methyl ester (1:5HFA ester) extracted from wasabi leaves can inhibit adipocyte differentiation and has anti-obesity and anti-diabetic functions in mice on a high-fat diet. The isosaponarin component in wasabi leaves can also promote collagen synthesis in fibroblasts.

[0005] However, there is still little research on the effects of wasabi leaves on cancer stem cell development. Therefore, this invention aims to find an effective method to inhibit the growth of stem cells in prostate cancer, ovarian cancer, breast cancer, lung cancer, and oral cancer by using a self-extracted wasabi leaf alcohol extract (EJ-1). The ultimate goal of this invention is to develop a wasabi-derived drug or health food that can inhibit cancer stem cell growth without toxicity to normal cells, and in this way, serve as a long-term health food or adjuvant therapy for cancer patients after treatment to reduce cancer recurrence. Summary of the Invention

[0006] Because the extract of this invention is obtained from complex natural raw materials through multiple processing steps, its composition and microstructure are extremely complex, making it difficult for existing analytical techniques to fully characterize it with a clear and complete chemical structural formula or the proportion of a single component. Therefore, this invention defines the extract through key preparation steps (such as solvent type, extraction conditions, purification sequence, etc.) to reflect the unique physicochemical properties and functions imparted to the final product by the preparation method. This format of defining the product based on the characteristics of the preparation method is intended to present the technical content of this invention clearly, completely, and accurately, and is not merely a limitation on the preparation method itself.

[0007] The singular terms “a,” “an,” and “the” used in this specification and the claims, unless otherwise stated, can refer to more than one object.

[0008] The words "or," "and," and "and" used in this specification, unless otherwise stated, refer to "or / and." Furthermore, the terms "comprising" and "including" are not restrictive open-ended conjunctions. The foregoing paragraphs are for systematic reference only and should not be construed as limiting the subject of the invention.

[0009] The terms "treatment," "for the treatment," and similar expressions are used herein to refer to the administration of a drug to achieve a certain effect. This effect is a therapeutic, partial, or complete cure of a disease and / or its symptoms. As used herein, "treatment" encompasses any treatment of cancer stem cell-related cancers in mammals (particularly humans) and includes: (a) suppressing the disease, i.e., halting its development; and (b) alleviating the disease, i.e., resolving the disease. In the treatment of tumors (e.g., tertiary breast cancer), therapeutic agents can directly reduce the growth and metastasis of tumor cells.

[0010] As used herein, the term “tumor” refers to the abnormal growth of cells or tissues and should be understood to include both benign (i.e., non-cancerous) and malignant (i.e., cancerous) growth. The term “tumor” refers to a tumor or something related to a tumor. The terms “antitumor agent” and “anticancer drug” should be understood to refer to substances that produce antitumor or anticancer effects in tissues, systems, animals, mammals, humans, or other subjects. The term “cancer stem cells (CSCs)” refers to cells with the capacity to initiate and maintain tumors, including the ability to proliferate extensively, form new tumors, and maintain cancer development; that is, cells with unlimited proliferative potential that drive this formation and tumor growth. CSCs are biologically distinct from bulk tumor cells and possess stem cell-related characteristics, particularly the ability to self-renew, proliferate, and generate all cell types in a given cancer sample. The term “cancer stem cells” or CSC includes genetic alterations in stem cells (SCs) and in cells that become CSCs.

[0011] Cancer stem cells (CSCs) are also known as tumor initiating cells, cancer stem-like cells, stem-like cancer cells, highly tumorigenic cells, tumor stem cells, solid tumor stem cells, or super malignant cells. On the other hand, there is also evidence that CSCs are fundamentally responsible for tumorigenesis, cancer metastasis, and cancer recurrence.

[0012] This invention provides a pharmaceutical composition comprising a wasabi leaf alcohol extract (EJ-1) and a pharmaceutically acceptable carrier, wherein the wasabi leaf alcohol extract is obtained by a method comprising the following steps: (a) providing a raw material comprising wasabi (Eutremajaponica) leaves and crushing the raw material to form an extract; (b) soaking the extract in 70-95% alcohol; and (c) concentrating the soaked extract under low pressure at 20-60°C to obtain the wasabi leaf alcohol extract; wherein the wasabi leaf alcohol extract is analyzed by UHPLC–ESI–Q-TOF-MS / MS, and the mass spectrometry results in positive ion mode are approximately 595.1657 [M+H] at m / z. + 449.1078[M+H] + 206.0667 [M+H] +It exhibits a characteristic ion peak at approximately 422.0243 [MH] in negative ion mode; and in this mode, it shows a peak at approximately [MH] m / z. - 358.0261 [MH] - And 431.0972 [MH] - Characteristic ion peaks are observed at these locations. These characteristic ion peaks collectively constitute the chemical fingerprint of this wasabi leaf alcohol extract.

[0013] In this invention, wasabi leaf alcohol extract can be used to inhibit the growth of cancer stem cells and effectively reduce the expression of cancer stem cell marker molecules, wherein the cancer stem cell marker molecules include CD133, ALDH1A1, CD44, Oct-4 and any combination thereof.

[0014] In one embodiment, the alcohol comprises ethanol.

[0015] In another embodiment, the cancer stem cells include: triple-negative breast cancer stem cells, ovarian cancer stem cells, prostate cancer stem cells, lung cancer stem cells, oral cancer stem cells, or human mesenchymal stem cells (hMSCs).

[0016] This invention provides the use of a pharmaceutical composition for preparing a medicament for treating cancer stem cell-related cancers, wherein the pharmaceutical composition comprises an alcoholic extract of wasabi (Eutrema japonica) leaves, wherein the cancer stem cell-related cancers include triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

[0017] In one embodiment, the cancer stem cells include triple-negative breast cancer stem cells, ovarian cancer stem cells, prostate cancer stem cells, lung cancer stem cells, oral cancer stem cells, or human mesenchymal stem cells (hMSCs).

[0018] In another embodiment, the wasabi leaf alcohol extract is administered to the individual with cancer via oral or intraperitoneal injection. In a preferred embodiment, the wasabi leaf alcohol extract is administered to the individual with cancer via oral administration.

[0019] In another embodiment, the effective dose is 50-750 mg / kg. In a preferred embodiment, the effective dose is 50-500 mg / kg.

[0020] In a preferred embodiment, when the wasabi leaf alcohol extract is administered by injection to the individual with cancer, the effective dose is 50-100 mg / kg.

[0021] In a preferred embodiment, when the wasabi leaf alcohol extract is administered orally to the individual with cancer, the effective dose is 500-750 mg / kg.

[0022] In one embodiment, the wasabi leaf alcohol extract is divided into multiple applications over a week or applied daily.

[0023] In this invention, the pharmaceutical composition is used for the manufacture of a medicament that inhibits the growth of cancer stem cells.

[0024] In another embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, comprising an oral carrier and a parenteral carrier, wherein the oral carrier comprises: a water-soluble or oil-soluble carrier selected from, but not limited to, water, ethanol, glycerol, propylene glycol, medium-chain triglycerides (MCT oil), or vegetable oils (such as olive oil or sunflower oil); and a solubilizer selected from, but not limited to, cyclodextrin (CD), polysorbate 80, and PEG. 400 or other suitable surfactants to improve the solubility of isothiocyanates (ITCs); solid carriers selected from, but not limited to, microcrystalline cellulose (MCC), sodium carboxymethyl cellulose (CMC-Na), or hydroxypropyl methyl cellulose (HPMC) as excipients for tablets or capsules; and microencapsulation and sustained-release technologies, wherein the wasabi leaf alcohol extract may be encapsulated in polylactic-co-glycolic acid copolymer (PLGA) microparticles, sodium alginate, or liposomes to improve intestinal absorption and drug stability; wherein the parenterally administered carrier comprises a sterile solution or suspension administered by subcutaneous or intraperitoneal injection, wherein the diluent for the sterile injectable solution or suspension comprises the following pharmaceutically acceptable water-soluble carriers: buffer selected from physiological saline (NS), phosphate-buffered saline (PBS), or Ringer's Solution; and solubilizer selected from PEG 400, Polysorbate, etc. 80; low concentration DMSO antioxidant, selected from vitamin C (ascorbic acid), vitamin E, glutathione (GSH); and nanocarriers for injectable formulations, which employ the following nanotechnology to enhance drug delivery efficiency and sustained-release effect: liposomes, solid lipid nanoparticles (SLN), nanoemulsions, or PLGA microspheres.

[0025] In another embodiment, the pharmaceutical composition further comprises a chemotherapeutic agent, wherein the chemotherapeutic agent comprises the group consisting of doxorubicin, taxanes, or combinations thereof, wherein the taxanes are docetaxel and paclitaxel.

[0026] The wasabi leaf extract of the present invention is used in combination with at least one chemotherapeutic agent to produce a synergistic antitumor effect that is superior to that when used alone.

[0027] The present invention further provides the use of a pharmaceutical composition for preparing a medicament to inhibit the recurrence or metastasis of cancer stem cells after cancer tumor resection, wherein the pharmaceutical composition comprises wasabi leaf alcohol extract, wherein the cancer includes triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

[0028] In one embodiment, the cancer stem cells include triple-negative breast cancer stem cells, ovarian cancer stem cells, prostate cancer stem cells, lung cancer stem cells, oral cancer stem cells, or human mesenchymal stem cells (hMSCs).

[0029] Since the self-renewal of cancer stem cells (CSCs) and the occurrence of tumors are closely related to the stimulation of angiogenesis, the present invention further provides the use of a pharmaceutical composition for preparing a drug that inhibits tumor-associated angiogenesis, wherein the pharmaceutical composition comprises the above-mentioned wasabi leaf alcohol extract.

[0030] In another embodiment, the effective dose is 50-750 mg / kg. In a preferred embodiment, the effective dose is 50-500 mg / kg.

[0031] When wasabi leaf alcohol extract is administered by injection to an individual with cancer, the effective dose is 50-100 mg / kg.

[0032] When wasabi leaf alcohol extract is administered orally to an individual with cancer, the effective dose is 500-750 mg / kg.

[0033] In one embodiment, the alcohol comprises ethanol.

[0034] The present invention also provides a method for treating cancer stem cell-related cancers, comprising administering an effective dose of wasabi leaf alcohol extract to a patient suffering from such cancer stem cell-related cancers, wherein such cancer stem cell-related cancers include triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

[0035] In one embodiment, the alcohol is ethanol.

[0036] In one embodiment, the cancer stem cells include triple-negative breast cancer stem cells, ovarian cancer stem cells, prostate cancer stem cells, lung cancer stem cells, oral cancer stem cells, or human mesenchymal stem cells (hMSCs).

[0037] In another embodiment, the effective dose is 50-750 mg / kg. In a preferred embodiment, the effective dose is 50-500 mg / kg.

[0038] The effective dose of the wasabi leaf alcohol extract of the present invention administered to the patient by injection is 50-100 mg / kg.

[0039] The effective dose of the wasabi leaf alcohol extract of the present invention for oral administration to the patient is 500-750 mg / kg.

[0040] The present invention also provides a method for inhibiting tumor-associated angiogenesis, comprising administering an effective dose of wasabi leaf alcohol extract to a patient suffering from cancer stem cell-associated cancer, wherein the cancer stem cell-associated cancer includes triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

[0041] The present invention further provides a method for inhibiting the recurrence or metastasis of cancer stem cells in patients after cancer tumor resection, comprising administering an effective dose of wasabi leaf alcohol extract to a patient suffering from cancer stem cell-related cancer, wherein the cancer stem cell-related cancer includes triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

[0042] The effective dose of the wasabi leaf alcohol extract of the present invention administered to the patient by injection is 50-100 mg / kg. Attached Figure Description

[0043] Figure 1 is a schematic diagram of the extraction process of wasabi leaf alcohol extract (EJ-1).

[0044] Figure 2 shows the chemical characterization and identification of representative compounds in the EJ-1 extract using UHPLC–ESI–Q-TOF-MS / MS.

[0045] Figure 3 shows the growth of human triple-negative breast cancer cells (Hs578T and Bt549) treated with EJ-1 (Figure 3A and Figure 3B), mouse triple-negative breast cancer cells (4T1) (Figure 3C), human prostate cancer cells (PC-3) (Figure 3D), human ovarian cancer cells (SKOV-I6) (Figure 3E) and OVS-1 (Figure 3F), human oral cancer cells (GCHNC9) (Figure 3G), mouse lung cancer cells (L1) (Figure 3H), human lung cancer cells (Brmx2) (Figure 3I), as well as normal human mammary epithelial cells (H184) (Figure 3J) and human mesenchymal stem cells (hMSCs) (Figure 3K).

[0046] Figure 4 shows a schematic diagram of cancer stem cell spheres after EJ-1 treatment, with Hs578T, SKOVI6, PC-3, CGHNC9 and L1 cells as representatives. As shown in the figure, compared with the control group, the spherical shape of cancer stem cells gradually decreased and even became fragmented as the EJ-1 concentration increased.

[0047] Figure 5 shows the statistical changes in sphericity of cancer stem cells after EJ-1 treatment. Nine cell lines—Hs578T (Figure 5A), Bt549 (Figure 5B), 4T1 (Figure 5C), SKOV-I6 (Figure 5D), OVS-1 (Figure 5E), GCHNC9 (Figure 5F), L1 (Figure 5G), Brmx2 (Figure 5H), and PC-3 (Figure 5I)—were treated with EJ-1 at concentrations ranging from 0.1 to 50 μg / mL, and the results were observed and recorded under a microscope after 96 hours. The quantitative statistical graph is the average (±SEM) of three independent experiments, with p-values ​​calculated using one-way ANOVA.

[0048] Figure 6 shows the protein expression of five cancer stem cell lines (Hs578T_CSC, 4T1_CSC, PC-3_CSC, SKOV-I6_CSC, and L1_CSC) after EJ-1 treatment, as well as the protein expression of these cancer stem cells. Proteins from Hs578T (Figure 6A), 4T1 (Figure 6B), PC-3 (Figure 6C), SKOV-I6 (Figure 6D), and L1 (Figure 6E) cancer stem cells without EJ-1 and from CSCs of each cancer type were collected (Figures 6F-6J) for Western blot analysis.

[0049] Figure 7 shows a schematic diagram of EJ-1 layering. Sample A of EJ-1 was initially divided into three blocks (①, ②, and ③), and samples B and C were collected separately using HPLC. Segments ① and ③ of sample B were collected, while segment ② of sample C was collected separately with 6-MSITC.

[0050] Figure 8 shows the inhibitory effect of different EJ-1 layers on cancer stem cells. The EJ-1 layers used in Figure 8A include ①, ②, and ③ as described in Figure 7. Figure 8B uses EJ-1 layers that include ① and ③ as described in Figure 7, but do not include segment ② of 6-MSITC.

[0051] Figure 9 shows the in vivo inhibitory effect of EJ-1 on tumors derived from breast cancer stem cells (BCSCs). Figure 9A is a schematic diagram of the animal experimental plan and drug concentration. Figure 9B shows the tumor growth curves of mice in the control group and the EJ-1 treatment group. Figure 9C shows the tumor weight statistics of mice in each group at the experimental endpoint. Figure 9D shows the flow cytometry analysis of cancer stem cell-related marker proteins (CD44+ / CD24+). Quantitative values ​​are expressed as mean ± standard error (mean ± SEM, n = 5). Statistical analysis was performed using one-way ANOVA; ns indicates not significant, *p < 0.05 and ***p < 0.001 indicate statistically significant differences.

[0052] Figure 10 shows the effect of EJ-1 in inhibiting tumor growth, cell proliferation, angiogenesis, and inflammatory cytokine production in a 4T1 breast cancer model. Figure 10A is a schematic diagram of the in vivo animal experiment design. 2.5 × 10 3Four T1 breast cancer cells were injected into the fourth pair of mammary fat pads of BALB / c mice (immunely healthy mouse model). One week after tumor establishment, mice were randomly assigned to receive either PBS (control group) or EJ-1 (50 mg / kg or 100 mg / kg). All treatments were administered via intraperitoneal injection (ip) three times a week for four weeks. Data are expressed as mean ± standard error (mean ± SEM) (n = 6 per group). Figure 10B shows the change in mouse body weight during treatment. Figure 10C shows the tumor growth curves of the control and EJ-1 treatment groups. Figure 10D shows the tumor weight measured at the experimental endpoint. Figure 10E shows representative immunohistochemical (IHC) staining images (200× magnification; scale bar: 50 μm) of Ki-67 expression in tumor tissues of the control and EJ-1 treatment groups, with quantitative analysis of Ki-67 positive areas on the right. Figure 10F shows representative immunohistochemical images of CD31 expression in tumor tissues of the control group and the EJ-1 treatment group (200× magnification; scale bar: 50 μm), with quantitative analysis of the CD31-positive region on the right. Figure 10G shows serum cytokine analysis. Concentrations of various immunoregulatory cytokines in mouse serum were measured, including IL-1α, IL-1β, IL-4, IL-17A, IL-10, GM-CSF, MCP-1, MIP-1β, and TNF-α. Data are expressed as mean ± standard error (mean ± SEM) (n = 6 per group). Tumor growth curves (C) were analyzed using two-way repeated-measures ANOVA with post-hoc analysis using the Sidak multiple comparison test; other comparisons were analyzed using one-way ANOVA with the Bonferroni post-hoc test. ns, no significant difference; *p<0.05; **p<0.01; ***p<0.001.

[0053] Figure 11 shows the inhibitory effect of EJ-1 on tumor growth in the Hs578T-FPI breast cancer model. Figure 11A is a schematic diagram of the in vivo animal experiment design. Hs578T-FPI human triple-negative breast cancer cells (1 × 10⁶ cells per mouse) were used. 6Tumor cells were injected into the fourth pair of mammary fat pads of SCID mice. Seven days after tumor implantation, mice were administered PBS or EJ-1 (50 or 75 mg / kg) intraperitoneally three times a week for four weeks. Figure 11B shows the changes in mouse body weight during the experiment (n=5 per group). Figure 11C shows the tumor growth curves of the control group and the EJ-1 treatment group. Figure 11D shows representative images of the resected tumors at the experimental endpoint and quantitative analysis of tumor weight. Data are expressed as mean ± standard error (mean ± SEM) (n=5). Statistical analysis was performed using one-way ANOVA with Bonferroni post-hoc test. *p<0.05 indicates statistical significance compared to the control group.

[0054] Figure 12 shows the reduction of primary lung cancer xenograft tumor volume by EJ-1. Figure 12A is a schematic diagram of the animal experimental plan and drug concentration. Figure 12B shows the changes in mouse body weight. Figure 12C shows the tumor growth curve of mice in the EJ-1 treatment group, compared with the control group. Figure 12D shows the tumor weight of mice in each group. Figure 12E shows the spleen weight of mice in each group. Statistical analysis was performed using Student's t-test, *p<0.05; **p<0.01; ***p<0.001. Figure 13 shows the inhibition of postoperative recurrence and lung metastasis by EJ-1 in an allogeneic breast cancer model. Figure 13A is a schematic diagram of the experimental design of the 4T1 / BALB / c triple-negative breast cancer (TNBC) postoperative recurrence model. 4T1-GFP-Luc cells (2.5×10⁻⁶) were used. 3(Number of mice) were injected into the fourth pair of mammary fat pads of BALB / c mice. When the in situ tumor grew to approximately 0.6 cm in diameter at week 4, tumor resection was performed. Postoperatively, mice were randomly assigned to groups and administered either PBS or EJ-1 (100 mg / kg) intraperitoneally three times a week for three weeks. Figure 13B shows the changes in mouse body weight during treatment. No significant difference in body weight was observed between the control group and the EJ-1-treated group, indicating that EJ-1 administration did not cause significant systemic toxicity. Figure 13C shows in vivo bioluminescent imaging (IVIS) of tumor recurrence and metastasis at weeks 1 and 3 postoperatively. The figure shows representative images; the right side shows quantitative analysis of photon flux, showing that the tumor burden in the EJ-1-treated group was significantly lower than that in the control group. Figure 13D shows representative gross images of the lungs obtained at the experimental endpoint, showing a significant reduction in the number of lung metastatic nodules in the EJ-1-treated group; the right side shows quantitative analysis of the number of lung metastatic nodules. Figure 13E shows representative hematoxylin-eosin (H&E) stained sections of liver and lung tissue from the control group and the EJ-1 treatment group, presented at 4× and 50× magnification, respectively. Tumor infiltration areas are indicated by arrows. Pathological scoring of metastatic lesions was performed semi-quantitatively based on the percentage of tumor infiltration area, with the following scoring criteria: 0, no metastatic lesions; 1, very few (<1%); 2, mild (1–25%); 3, moderate (26–50%); 4, moderate to severe (51–75%); 5, severe (76–100%). Data are expressed as mean ± standard error (mean ± SEM) (n = 6). Statistical analysis was performed using one-way ANOVA with Bonferroni post-hoc test. *p < 0.05; **p < 0.01.

[0055] Figure 14 illustrates the enhancement of doxorubicin's antitumor efficacy in a syngeneic mouse TNBC model. Figure 14A is a schematic diagram of the treatment process. 2.5 × 10 3Four T1 breast cancer cells were injected into the fourth pair of mammary fat pads of BALB / c mice, followed by treatment with PBS (control group), EJ-1 (100 mg / kg, intraperitoneal injection, three times a week), doxorubicin (DXR; 8 mg / kg, intraperitoneal injection, once a week), or a combination of EJ-1 and DXR, for a period of four weeks. Figure 14B shows the weekly monitoring of mouse body weight during treatment; no significant differences were observed between the groups. Figure 14C shows the tumor growth curve during the four-week treatment period. Compared with the control group or the single-drug treatment group, the combination treatment group significantly inhibited tumor growth. Figure 14D shows representative images of the resected tumor at the experimental endpoint. Figure 14E shows the quantitative analysis of tumor weight at the experimental endpoint. The tumor weight in the combination treatment group was significantly smaller than that in the control group and the single-drug treatment group. Tumor growth inhibition (TGI) was calculated based on the mean tumor weight at the experimental endpoint. Figure 14F shows a representative hematoxylin-eosin (H&E) staining image of liver tissue, illustrating tumor infiltration (left: 20×; right: 100×). Figure 14G shows the histopathological scoring of metastatic liver lesions. Lesions were semi-quantitatively graded from 0 to 5 based on tumor infiltration area: 0, no lesions; 1, very few (<1%); 2, mild (1–25%); 3, moderate (26–50%); 4, moderate to severe (51–75%); 5, severe (76–100%). Data are expressed as mean ± standard error (mean ± SEM) (n = 6 per group). Tumor growth curves (C) were analyzed using two-way repeated-measures ANOVA with Sidak multiple comparison test; other data were analyzed using one-way ANOVA and Bonferroni post-hoc test. ns, not significant; *p<0.05; **p<0.01; ***p<0.001.

[0056] Figure 15 shows the inhibitory effect of different concentrations of EJ-1 on angiogenesis in human umbilical vein endothelial cells (HUVECs). Figure 15A shows representative images of lumen formation in HUVECs after treatment with different conditioned media for 48 hours. Conditioned media sources included: no treatment control, Avastin (10 μg / mL), Cyramza (10 μg / mL), EJ-1 (1 μg / mL), EJ-1 (10 μg / mL), and DMSO (0.015%). The EJ-1 group, especially 10 μg / mL, showed a significant reduction in tubular networks. Scalebar = 200 μm. Figure 15B shows the quantitative results of node number, presented as a percentage of the control group. Figure 15C shows the quantitative results of grid number, presented as a percentage of the control group. Data are expressed as mean ± standard deviation. Statistical significance: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Detailed Implementation

[0057] Various embodiments of the invention are discussed in more detail below. However, these embodiments may be specific to various applications of the inventive concept and can be practiced in various specific environments. These embodiments are for illustrative purposes only and do not limit the scope of this disclosure.

[0058] Example 1: Cell line origin and enrichment method for breast cancer stem cells

[0059] Human breast cancer cells Hs578T, mouse breast cancer cells 4T1, and normal breast cells H184 were obtained from ATCC. Mouse lung cancer cells L1, prostate cancer cells PC-3, human ovarian cancer cells SKOV-I6 and OVS-1, and human oral cancer cells GCHNC9 were established in this experiment. Human lung cancer cells Brmx2 were donated by Professor She Yuping of our school, and human mesenchymal stem cells (hMSCs) were donated by Professor Hong Shijie of our school. All cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS and 1% penicillin and streptomycin sulfate at 37°C and 5% CO2. When the cells expanded to approximately 80% confluence with the culture dish, subculturing was performed. The old culture medium was removed, and the cells were washed with PBS. After washing, trypsin-EDTA was added and incubated at 37°C for approximately 5 minutes. The trypsin-EDTA effect was then terminated with serum-containing culture medium, and the cells were simultaneously rinsed off. An appropriate amount of cell culture was then transferred to a new culture dish, and serum-containing culture medium was added for further culture. To enrich cancer stem cells (CSCs), 1x102 cells were cultured. 4 Parental cells of Hs578T, 4T1, L1, PC-3, SKOV-I6, or OVS-1 were seeded in Basal StemFlex Medium and treated with 1% penicillin-streptomycin. Cells were cultured in low-adhesion 10 cm medium at 37°C with high humidity (5% CO2) for 8 days. Cancer stem cells (CD44+ / CD133+) were then enriched using a cell sorting system.

[0060] Example 2: Preparation of Wasabi Leaf Alcohol Extract (EJ-1)

[0061] Fresh wasabi leaves were sourced from Flower Biotechnology Co., Ltd. in Taiwan, China. As shown in Figure 1, fresh wasabi leaves were extracted using a ratio of 600g fresh leaves to 2L of 95% ethanol. The leaves were soaked at room temperature for 12 hours to recover the extract, and then soaked again with an equal amount of 95% ethanol for another 12 hours, for a total of 24 hours. All extract was then recovered and filtered. The next step was low-pressure concentration at 40°C, followed by low-temperature vacuum freeze-drying to turn the extract into powder, which was then stored at -80°C.

[0062] Example 3: Chemical Composition Analysis of EJ-1

[0063] To ensure the chemical identifiability of the EJ-1 extract, this invention employs ultra-high performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS / MS) for chemical fingerprint analysis, with the assistance of the Proteomics Core Facility. The EJ-1 extract was analyzed in both positive and negative ion free modes, and the resulting base peak chromatograms (BPCs) are shown in Figure 2. The selected quality control (QC) markers collectively represent the major chemical classes present in wasabi leaves, including flavonoid C-glycosides (isosaponins, isovitexin, and glucosinolates), glucosinolates (glucosinolates and glucosyl isothiocyanates), and the bioactive isothiocyanate derivative 6-methylsulfinylhexyl isothiocyanate. These markers collectively represent the major phytochemical categories naturally present in wasabi leaves. By comparing their retention time, precise mass, and MS / MS fragmentation patterns with control standards, six representative compounds were identified, as shown in Figure 2. In positive ion mode, isosaponarin (peak 1), cynaroside (peak 2), and 6-methylsulfinylhexyl isothiocyanate (6-MSITC, peak 3) were identified; in negative ion mode, glucoiberin (peak 4), sinigrin (peak 5), and isovitexin (peak 6) were identified. These compounds represent the major phytochemical categories commonly found in wasabi leaves, including flavonoids, glucosinolates, and isothiocyanate derivatives. By comparing the retention time, precise mass, and MS / MS fragmentation spectra of the standards, six representative compounds were identified. The corresponding MS / MS fragmentation spectra of each compound are also presented, along with their characteristic fragment ions.

[0064] Furthermore, this invention utilizes external calibration standards for quantitative analysis to determine the content of the aforementioned six compounds in the EJ-1 extract, and the results are summarized in Table 1. Overall, the data show that the EJ-1 extract has a well-defined and reproducible chemical composition.

[0065] Table 1. Identification and quantification of representative compounds in EJ-1 extracts

[0066] Example 4: Inhibition of cancer cell proliferation by EJ-1

[0067] Cell proliferation was detected using the MTS Assay kit (Abcam). The assay was performed according to the manufacturer's instructions. 3 × 10⁻⁶ cells were added to each 96-well plate. 3 Cells were cultured, and different concentrations of EJ-1 extract were added every other day. Measurements were performed at 24 and 48 hours. Before measurement, the liquid in the 96-well plate was aspirated, and MTS was mixed with cell culture medium at a ratio of 1:4. 100 μl of the mixture was added to each well of the 96-well plate and reacted at 37 °C for 1.5 hours. The amount of MTS, which is proportional to the number of viable cells, was measured at 490 nm using a 96-well plate reader. Each experiment was repeated three times, and the data shown are mean ± SEM.

[0068] To evaluate whether EJ-1 inhibits the growth of human triple-negative breast cancer cells (Hs578T and Bt549), mouse breast cancer cells (4T1), human prostate cancer cells (PC-3), human ovarian cancer cells (SKOV-I6 and OVS1), human oral cancer cells (GCHNC9), mouse lung cancer cells (L1), and human lung cancer cells (Brmx2), Figure 2 shows the half-maximal inhibitory concentration (IC50) of EJ-1 in the nine cancer cell lines compared to the untreated control group using different concentrations of EJ-1. 50 The concentrations of the active ingredients (Hs578T, 271.3 μg / mL, Figure 3A), Bt549, 4T1, PC-3, SKOV-I6, OVS-1, GCHNC9, L1, and Brmx2 were 169.2 μg / mL (Figure 3I). This demonstrates that the inhibition of cell growth is dose-dependent and cell-specific, with EJ-1 achieving the greatest cell growth inhibition (>80%) at a concentration of 500 μg / mL.

[0069] In addition, EJ-1 was used to treat normal cells and stem cells, and the IC50 of these two cell lines and various cancer cells treated with EJ-1 was compared. 50 The difference was observed in the results, showing that normal mammary epithelial cells-H184 treated with EJ-1 had reduced IC50. 50 The concentration of human mesenchymal stem cells (hMSCs) treated with EJ-1 was 454.4 μg / mL (Figure 3J), and its IC50 concentration was 454.4 μg / mL (Figure 3J). 50 The concentration was 417.7 μg / mL (Figure 3K). The IC50 of these two cell lines was...50 All were higher than the IC50 of EJ-1 in treating various cancer cell types. 50 Therefore, the results above show that EJ-1 is toxic to cancer cells, but less toxic to normal cells or mesenchymal stem cells, as shown in Table 2.

[0070] Table 2. Comparison of IC50 values ​​for monolayer cell growth assay of various cancer cells after EJ-1 treatment. 50 Sorting Table

[0071] Example 5: EJ-1's ability to inhibit cancer stem cell growth

[0072] First, a sphere formation assay is performed on cancer stem cells, with 5 x 10⁸ cells of each type seeded. 4 Cells were cultured in 24-well ultra-low attachment (ULA) plates using Basal StemFlex Medium. Experiments were performed using EJ-1 at concentrations of 1, 5, 10, 25, or 50 μg / mL. After 96 hours of culture, images were taken using the Lionheart LX automated live-cell imaging system (BioTek), and the number of spheroids formed was counted. Data are presented as mean ± SEM; all experiments were performed three times.

[0073] To investigate whether EJ-1 can inhibit the growth of cancer stem cells, cancer stem cells were isolated from cancer cells using a special culture medium (3D-sphere culture assay), followed by a sphere formation assay to confirm EJ-1's ability to inhibit cancer stem cell growth. Cell morphology results showed that cancer stem cells treated with a low concentration of EJ-1 (0.1 μg / mL) did not exhibit significant changes compared to the control group. However, with increasing concentration, the spheres became smaller and even ruptured, as shown in Figure 4. This indicates that at higher concentrations, EJ-1 has a significant inhibitory effect on spherical cancer stem cells.

[0074] Figure 5 shows the statistical results of the IC50 values ​​of two human triple-negative breast cancer cells (Hs578T and Bt549), mouse triple-negative breast cancer (4T1), prostate cancer PC-3, ovarian cancer (SKOV-I6 and OVS-1), oral cancer (GCHNC9), and lung cancer (L1 and Brmx2) treated with EJ-1. 50 The concentrations were 4.7, 1, 10, 4.5, 4.9, 7.1, 1, 26.8, and 7 μg / mL, respectively. The results of the previous two experiments are combined and shown in Table 2, comparing the IC50 of EJ-1 in cancer cells and cancer stem cells.50 The IC50 of EJ-1-treated spherical cancer stem cells was found to be high. 50 The difference was 5 to 197 times lower than that of cancer cells growing in a single layer. Therefore, these phenomena suggest that EJ-1 may be more specific to cancer stem cells.

[0075] The results in Table 3 show that EJ-1 is more specific to cancer stem cells and less toxic to normal cells and mesenchymal stem cells.

[0076] Table 3. IC50 values ​​of EJ-1 in cell proliferation assays and cancer stem cell spheroidization assays for breast cancer, ovarian cancer, prostate cancer, oral cancer, lung cancer cells, normal cells, and mesenchymal stem cells. 50 Summary table.

[0077] Example 6: Expression Analysis of Cancer Stem Cell Marker Molecular Proteins

[0078] After washing the cells with PBS, RIPA cell lysis buffer (pH 7.4) containing a protease inhibitor cocktail was added to scrape the cells. After centrifugation, the supernatant was collected, and the protein was quantified using the Coomassieplus protein assay kit. An equal volume of cell protein was mixed with Laemmlis sample buffer and heated at 98°C for 10 minutes.

[0079] Samples were injected into the SDS-PAGE sample well for electrophoretic separation. After electrophoresis, the separation colloid was carefully removed, and the proteins on the colloid were transferred to a PVDF membrane. The target protein locations were determined by staining with Ponceau S solution. The transfer mold at the appropriate location was cut off and washed three times with washing buffer (pH 7.4) containing 0.1% Tween-20. Then, the membrane was reacted sequentially with buffer (pH 7.4) containing 5% skim milk powder, the primary antibody, and the corresponding secondary antibody. Finally, the protein was developed using an Enhanced Chemiluminescence (ECL) developing kit and analyzed using a ChemiDoc XRS+ digital imaging system.

[0080] The previous cell experiments showed that EJ-1 is specific for inhibiting the growth of cancer stem cells. Therefore, to further understand whether EJ-1 has a direct effect on cancer stem cell marker molecules, we used the previously described spheroidization assay to measure the IC50 of various cancer stem cells for EJ-1. 50Subsequently, Western blot analysis was performed. As shown in Figure 5, the protein expression of five cancer stem cell lines (breast cancer cells (Hs578T_CSC, 4T1_CSC), prostate cancer cells (PC-3_CSC), ovarian cancer cells (SKOV-I6_CSC), and lung cancer cells (L1_CSC)) was identified, and the differences in the expression of cancer stem cell marker molecules between the original cancer cells (parental) and the enriched cancer stem cells were compared.

[0081] The results showed that, compared with the parental control group, enriched Hs578T, 4T1, and PC-3 cancer stem cells (CSCs) induced increased expression of cancer stem cell markers CD133, ALDH1A1, CD44, and Oct-4 (Figures 6A, 6B, and 6C). SKOV-I6 cancer stem cell markers also induced greater expression of CD133, ALDH1A1, and CD44 in enriched CSCs (Figure 6D), and L1 cancer stem cell markers induced expression of CD133, CD44, and Oct-4 proteins in enriched CSCs (Figure 6E). Therefore, these results demonstrate that this embodiment successfully enriched the expression of cancer stem cells from various cancer types.

[0082] Next, the expression of cancer stem cell marker proteins was compared among cancer stem cells of different cancer types after 48 hours of EJ-1 treatment. Results showed that after EJ-1 treatment, the protein expression of CD133, ALDH1A1, CD44, and Oct-4 decreased in Hs578T_CSC, 4T1_CSC, PC-3_CSC, and SKOV-I6_CSC (Figures 6F, 6G, 6H, and 6I). In contrast, EJ-1 treatment of L1_CSC showed a trend of reduced expression of CD133, CD44, and ALDH1A1 (Figure 6J).

[0083] In summary, the results show that enriched cancer stem cells can indeed induce the expression of more cancer stem cell marker molecules; and EJ-1 treatment can further reduce the expression of cancer stem cell marker molecules in breast cancer, ovarian cancer, prostate cancer, and lung cancer cells.

[0084] Example 7: Stratified Analysis of Active Ingredients in EJ-1

[0085] As shown in Figure 7, sample A (EJ-1) was initially divided into three blocks (①, ②, and ③), and samples B and C were collected using HPLC. Sample B only had blocks ① and ③ collected, primarily to remove 6-MSITC from EJ-1, while sample C contained block ②, which simply retained 6-MSITC. Next, cell experiments were conducted on samples A and B to analyze whether 6-MSITC was the main reason for EJ-1's ability to inhibit cancer stem cells.

[0086] As shown in Figure 8, the IC50 of the original EJ-1 (Figure 8A) and EJ-1 with 6-MSITC removed (Figure 8B) for cancer stem cell inhibition are compared. 50 Data showed that the presence or absence of 6-MSITC did not significantly affect the ability of EJ-1 to inhibit cancer stem cells. Therefore, 6-MSITC is not the main active ingredient in this extract that inhibits cancer stem cells.

[0087] Example 8: In vivo inhibition of breast cancer stem cell (BCSC)-derived tumors by EJ-1

[0088] To investigate the effect of EJ-1 on tumor growth induced by cancer stem cells, 4T1 cancer cells and enriched 4T1-CSC cancer stem cells (characterized by CD44) were separately cultured. + / CD24 - / CD133 + Take 1x10 2 Cell solution and basement membrane matrix ( Basement Matrix (EMM) was mixed in a 7:3 ratio and injected orally into the fourth pair of mammary fat pads of 6-8 week old female BALB / c mice. 4T1 and 4T1-CSC cells were implanted, and after one week of tumor formation, drug injection began. EJ-1 was administered intraperitoneally at a concentration of 100 mg / kg, three times a week for five weeks, as shown in the flowchart in Figure 9A. Mouse body weight and tumor size were measured weekly. Tumor volume was calculated using the following formula: Tumor volume (mm²) 3 = [Length (mm) × Width (mm)] 2[×0.5] At the end of the experiment, mice were sacrificed using CO2, and tumors and organ tissues (heart, liver, spleen, lungs, and kidneys) were removed. After removal, the tumor and spleen tissues were imaged and their weight measured. The tumor tissue was divided into three equal parts. The first part was dissociated into a single-cell suspension using a mouse tumor dissociation kit, and analyzed by flow cytometry after density gradient centrifugation. The second part was stored at -80°C and thawed for further protein extraction and analysis. The third part of the tumor tissue, along with other organ tissues, was fixed in 10% formalin and sent to the Animal Medical Testing Center of National Chung Hsing University for slide preparation and H&E staining. The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC).

[0089] As shown in Figure 9B, EJ-1 significantly inhibited tumor growth in both the 4T1 and 4T1-CSC tumor models. Notably, EJ-1 exhibited a more pronounced inhibitory effect on tumors derived from CSC-enriched cells (4T1-CSC). At the experimental endpoint of tumor weight analysis, EJ-1 reduced the tumor weight of 4T1 tumors by approximately 40%, while in CSC-derived tumors, the tumor weight was significantly reduced by approximately 76%, as shown in Figures 9C and 9D. This result indicates that tumors initiated by CSC-enriched populations are highly sensitive to EJ-1 treatment in vivo.

[0090] To further evaluate the effect of EJ-1 on CSC-related populations in tumors, flow cytometry analysis was performed on dissociated tumor tissues. The results showed that EJ-1 treatment significantly reduced CD44 levels in tumors formed from 4T1-CSC cells. + / CD24 - The proportion of cells is shown in Figure 9D; in contrast, only slight changes in the CSC-like cell population were observed after EJ-1 treatment in parental 4T1 tumors. Based on the above in vivo experimental results, EJ-1 not only effectively inhibits tumor growth but also preferentially reduces the CSC-related cell population in CSC-driven tumors, supporting its role in inhibiting tumor stemness and CSC-related tumorigenesis in vivo.

[0091] These results indicate that EJ-1 can effectively target BCSCs and inhibit their tumorigenesis, supporting its potential application as a candidate drug for anticancer treatment.

[0092] Example 9: Antitumor effect of EJ-1 in allogeneic / xenograft models of triple-negative breast cancer (TNBC)

[0093] First, the antitumor effect of EJ-1 was evaluated in a 4T1 syngeneic triple-negative breast cancer (TNBC) mouse model (with a complete immune system). Mice were injected with 2.5 × 10⁻⁶ oz. fat pads in the fourth pair of mammary glands. 3 4 T1 cells were implanted. One week after tumor implantation, mice were administered either the vector control or EJ-1 (50 or 100 mg / kg) via intraperitoneal injection three times a week, as shown in Figure 10A. Throughout the experiment, the body weight of each treatment group remained stable (Figure 10B), indicating that EJ-1 was well tolerated at both doses. EJ-1 inhibited tumor growth in a dose-dependent manner, with the 100 mg / kg group showing a more significant inhibitory effect from the third week onwards (Figure 10C). Tumors collected at the experimental endpoint showed that the tumor volume (Figure 10C) and tumor weight (Figure 10D) of the 100 mg / kg group were significantly lower than those of the control group.

[0094] To further characterize the antitumor effect of EJ-1, immunohistochemical (IHC) analysis was performed on tumor tissues from the 100 mg / kg group to assess indicators related to tumor proliferation and angiogenesis. IHC staining results showed that EJ-1 significantly reduced the number of Ki-67-positive tumor cells (Figure 10E), indicating that tumor cell proliferation was inhibited. Furthermore, EJ-1 also significantly reduced the density of CD31-positive microvessels in tumor tissues (Figure 10F), demonstrating its inhibitory effect on tumor-associated angiogenesis.

[0095] Furthermore, to assess whether EJ-1 affects systemic inflammatory responses, the concentrations of cytokines in the serum of tumor-bearing mice were measured. EJ-1 treatment significantly reduced multiple pro-tumor and immunosuppressive cytokines (Figure 10G and H), including IL-1α, IL-1β, IL-4, IL-10, MCP-1, and GM-CSF. These results suggest that the anti-tumor effect of EJ-1 may be partly due to the suppression of systemic inflammatory responses, thereby reducing the formation of an immunosuppressive tumor microenvironment within the tumor.

[0096] To further evaluate the antitumor effect of EJ-1 in a human TNBC model, Hs578T-FPI human TNBC cells were implanted into the fourth pair of mammary fat pads of SCID mice. Mice were injected with 1×10-1 6Hs578T-FPI cells were injected intraperitoneally with either the vector control or EJ-1 (50 or 75 mg / kg) three times a week after tumor implantation, as shown in Figure 11A. EJ-1 also significantly inhibited tumor growth in this xenograft model, as evidenced by a significant reduction in tumor volume and weight compared to the control group (Figures 11C and 11D). No significant difference in tumor inhibition was observed between the two EJ-1 dosage groups. Furthermore, EJ-1 treatment did not cause significant changes in body weight (Figure 11B). In summary, EJ-1 effectively inhibits tumor growth, and this effect is applicable to both immunocompetent and immunodeficient TNBC mouse models.

[0097] Example 10: EJ-1 inhibits tumor growth in an immunocompetent mouse lung cancer model

[0098] To establish an animal model of lung cancer tumor suppression, as shown in Figure 12A, mouse lung cancer cells (L1) were injected subcutaneously into the left lower back of immunocompetent female BALB / c mice. After tumor formation, EJ-1 was administered intraperitoneally at doses of 75 and 100 mg / kg. Mouse body weight was recorded weekly, and weight changes are shown in Figure 12B. No significant changes in body weight were observed throughout the experiment, with all animals experiencing weight changes within 10%. As shown in Figure 12C, EJ-1 treatment showed a significant trend towards inhibiting tumor growth compared to the control group. As shown in Figure 12D, the tumor weight results indicated a more significant inhibitory effect in the group receiving 100 mg / kg EJ-1. As shown in Figure 12E, there was no significant difference in spleen weight between the EJ-1-treated groups and the control group. In conclusion, EJ-1 also exhibits inhibitory effects against lung cancer.

[0099] Example 11: EJ-1 inhibits spontaneous metastasis after surgery

[0100] To evaluate the potential of EJ-1 as a post-treatment adjuvant therapy, a TNBC model based on 4T1-GFP-Luc mouse triple-negative breast cancer cells was established in immunocompetent female BALB / c mice. 2.5 × 10⁻⁶ cells were used. 3 Four T1-GFP-Luc cells were injected into the fourth pair of mammary fat pads. When the tumor grew to approximately 0.6 mm in diameter (approximately 4 weeks post-implantation), in situ tumor resection was performed. Three days post-surgery, mice were administered either PBS or EJ-1 (100 mg / kg) via intraperitoneal injection (Figure 13A). Serial IVIS in vivo imaging analysis showed that control mice exhibited a significantly increased metastatic tumor burden within three weeks post-surgery; in contrast, mice in the EJ-1 treatment group showed a significantly reduced bioluminescent signal intensity, indicating effective inhibition of spontaneous metastatic progression (Figure 13C).

[0101] Further gross observations revealed a significantly lower number of pulmonary metastatic nodules in the EJ-1-treated group compared to the control group (Figure 13D). Consistent with this, histopathological analysis of liver and lung tissues also showed a significant reduction in metastatic lesion scores in the EJ-1-treated group (Figure 13E). Throughout the treatment period, the body weight of mice in both groups remained stable, indicating that EJ-1 administration did not cause observable systemic toxicity (Figure 13B). In summary, EJ-1 effectively inhibited postoperative spontaneous metastasis in this TNBC model, supporting its potential as a postoperative adjuvant therapy strategy to limit cancer stem cell (CSC)-driven metastatic tumor regeneration and spread.

[0102] Example 12: EJ-1 enhances the efficacy of chemotherapy in in vivo experiments

[0103] To further explore the therapeutic potential of EJ-1, the antitumor effects of EJ-1 alone or in combination with chemotherapy drugs were evaluated by implanting 4T1 triple-negative breast cancer (TNBC) cells into the fourth pair of mammary fat pads of BALB / c mice. Mice received intraperitoneal injections of PBS, EJ-1 (100 mg / kg, three times a week), doxorubicin (DXR; 8 mg / kg, once a week), or a combination of EJ-1 and DXR for four weeks (Figure 14A). Throughout the experiment, the body weight of mice in all treatment groups remained stable (Figure 14B), showing no significant treatment-related weight loss.

[0104] Compared with monotherapy, the combination therapy group significantly inhibited tumor growth, as evidenced by a marked decrease in tumor volume over time (Figure 14C) and a higher tumor growth inhibition (TGI) rate of 84% calculated based on the tumor weight at the experimental endpoint (Figures 14D and 14E), as shown in Table 4. Furthermore, the tumor weight analysis at the experimental endpoint showed a significant difference between the combination therapy group and the EJ-1 monotherapy group (Figure 14E), indicating that the combination of EJ-1 and doxorubicin further enhances the therapeutic effect. On the other hand, histopathological analysis showed that the combination therapy significantly reduced the area of ​​tumor-infiltrating lesions in the liver (Figures 14F and 14G).

[0105] Table 4. Tumor growth inhibition rate under different treatment groups

[0106] In summary, EJ-1 can enhance the antitumor efficacy of doxorubicin without causing significant weight loss, demonstrating its potential as an adjuvant therapy strategy to inhibit tumor progression and metastatic tumor burden in TNBC.

[0107] Example 13: EJ-1 inhibits angiogenesis in human umbilical vein endothelial cells (HUVECs)

[0108] Cancer stem cell (CSC) self-renewal and tumorigenesis are closely related to angiogenesis, which is achieved through the release of pro-angiogenic substances such as vascular endothelial growth factor (VEGF). Previous studies have shown that EJ-1 can inhibit key signaling proteins such as VEGFR, PI3K, AKT, and mTOR. Therefore, this study further evaluated whether EJ-1 affects the pro-angiogenic capacity of TNBC cells. To assess whether EJ-1 has anti-angiogenic capabilities, this study used green fluorescent human umbilical vein endothelial cells (GFP-HUVECs) for an in vitro tube formation assay, while treating GFP-HUVECs with different concentrations of EJ-1.

[0109] This embodiment follows the experimental method of Du et al. (2020) to conduct a tubulogenesis experiment of HUVECs in “Du P, Subbiah R, Park JH, Park K. Vascular morphogenesis of human umbilical vein endothelial cells on cell-derived macromolecular matrix microenvironment. Tissue Eng Part A. 2014 Sep; 20(17-18):”. The ibidiμ-Slide 15 Well 3D was used, and the HUVEC tubulogenesis experiment was performed according to the manufacturer's instructions. First, a thin layer of matrigel was pre-added to each well and cured at 37°C for 30 minutes. Then, HUVECs were placed at 2 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μg / mL in the central wells and allowed to stand for 3 hours to allow for cell attachment. After cell attachment, conditioned medium at a concentration of 100 μg / mL was added to each well. This conditioned medium was derived from cancer cell culture media that had undergone different treatment conditions.

[0110] To assess anti-angiogenic activity, Avastin (10 μg / mL) or Cyramza (10 μg / mL) was added to the conditioned medium of the control group; the effect of EJ-1 was tested using conditioned medium collected from cancer cells treated with EJ-1 at 1 μg / mL or 10 μg / mL. A solvent control group containing 0.015% DMSO was also included in the experiment.

[0111] HUVECs were incubated with each group of conditions for 48 hours to form a vascular network, and stained with acridine orange for observation. Images were captured using the BioTek Lionheart LX automated live-cell imaging system. Angiogenesis parameters, including the number of nodes and the number of grids, were quantified using ImageJ's Angiogenesis Analyzer.

[0112] As shown in Figure 15A, the conditioned medium obtained after EJ-1 treatment significantly reduced the formation of the luminal network in HUVECs in a concentration-dependent manner. Quantitative analysis results are shown in Figures 15B and 15C, indicating a significant decrease in both the number of nodes and the number of grids. Notably, at a concentration of 10 μg / mL, the inhibitory effect of EJ-1 was similar to that of Cyramza and significantly stronger than that of Avastin.

[0113] In summary, EJ-1 can reduce the pro-angiogenic capacity of TNBC cells, suggesting that its inhibition of angiogenesis may be one of the important contributions to its overall anti-tumor effect.

[0114] While the invention has been described and illustrated in sufficient detail to enable those skilled in the art to make and use it, various alternatives, modifications and improvements should be apparent without departing from the spirit and scope of the invention.

[0115] Those skilled in the art will readily understand that the present invention is highly suitable for achieving the above-described objects and obtaining the aforementioned objects and advantages, as well as those inherent therein. The processes and methods described above for producing them represent preferred embodiments, are exemplary, and do not limit the scope of the invention. Modifications and other uses will be apparent to those skilled in the art. These modifications are contained within the spirit of the invention and are defined by the scope of the claims.

Claims

1. Use of a pharmaceutical composition for the preparation of a medicament for treating cancer stem cell-related cancers, wherein the pharmaceutical composition comprises a wasabi leaf alcohol extract. The cancer stem cell-related cancers mentioned above include triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

2. The use as described in claim 1, wherein the alcohol is ethanol.

3. The use as described in claim 1, wherein the cancer stem cells are triple-negative breast cancer stem cells, ovarian cancer stem cells, prostate cancer stem cells, lung cancer stem cells, oral cancer stem cells, or human mesenchymal stem cells.

4. The use as described in claim 1, wherein the effective dose of the wasabi leaf alcohol extract is 50-750 mg / kg.

5. The use as claimed in claim 1, wherein the pharmaceutical composition further comprises a chemotherapeutic agent.

6. The use as claimed in claim 5, wherein the chemotherapeutic agent is a group consisting of doxorubicin, taxanes, or combinations thereof.

7. The use as claimed in claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8. Use of a pharmaceutical composition for preparing a medicament to inhibit recurrence or metastasis of cancer stem cells after tumor resection, wherein the pharmaceutical composition comprises a wasabi leaf alcohol extract. The cancers mentioned include triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

9. Use of a pharmaceutical composition for preparing a medicament that inhibits tumor-associated angiogenesis, wherein the pharmaceutical composition comprises a wasabi leaf alcohol extract.

10. A method of treating cancer stem cell-related cancers, comprising administering an effective dose of wasabi leaf alcohol extract to a patient suffering from said cancer stem cell-related cancers, wherein said cancer stem cell-related cancers include triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

11. The method of claim 10, wherein the cancer stem cells comprise triple-negative breast cancer stem cells, ovarian cancer stem cells, prostate cancer stem cells, lung cancer stem cells, oral cancer stem cells, or human mesenchymal stem cells.

12. The method of claim 10, wherein the effective dose is 50-750 mg / kg.

13. A method for inhibiting tumor-associated angiogenesis, comprising administering an effective dose of wasabi leaf alcohol extract to a patient suffering from said cancer stem cell-associated cancer, wherein said cancer stem cell-associated cancer includes triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

14. A method for inhibiting recurrence or metastasis of cancer stem cells in a patient after cancer tumor resection, comprising administering an effective dose of wasabi leaf alcohol extract to a patient suffering from said cancer stem cell-related cancer, wherein said cancer stem cell-related cancer includes triple-negative breast cancer, ovarian cancer, prostate cancer, lung cancer, or oral cancer.

15. The method of claim 13, wherein the effective dose is 50-100 mg / kg.

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