KR102185544B1 - Composition for treating thyroid carcinoma containing evodiamine and chemotherapeutic agent - Google Patents

Abstract

Background/Purpose: The purpose of this study is to evaluate the effect of evodiamine in combination with chemotherapy on thyroid cancer cells. Materials and Methods: TPC-1 and SW1736 thyroid cancer cells were used. Cell viability, cytotoxic activity, cell death and migration were investigated by applying appropriate methods. Drug combination analysis was performed. RESULTS: When evodiamine was treated, cell viability, Bcl2 and phosphorylated-AKT protein levels decreased, and cytotoxicity and the percentage of apoptotic cells increased. When treated with wortmannin, cell viability and phosphorylated-AKT and Bcl2 protein levels decreased, and cytotoxic activity increased. In TGF-β-treated cells, evodiamine attenuated changes in morphology, growth, and migration, increased p21 and p53 protein levels, and β-catenin, N-cadherin, vimentin, phosphorylated-AKT, MMP-2 And decreased MMP-9 protein levels. When evodiamine and chemotherapeutic were treated together, the combination index was less than 1.0. CONCLUSION: Evodiamine showed cytotoxicity in thyroid cancer cells, and inhibition of AKT enhanced evodiamine-induced cytotoxicity. In addition, evodiamine inhibited the proliferation, migration, and epithelial-mesenchymal metastasis of thyroid cancer cells, and synergized with chemotherapeutic agents.

Description

Composition for treating thyroid carcinoma containing evodiamine and chemotherapeutic agent}

The present invention relates to a composition for the treatment of thyroid cancer comprising evodiamine and a chemotherapeutic agent, and evodiamine alone caused apoptosis with co-mutation of a survival-related protein, and inactivation of AKT caused evodiamine to survive thyroid cancer cells. The negative effect was reinforced. In addition, evodiamine attenuated proliferation, migration and epithelial-mesenchymal metastasis by regulating epithelial-mesenchymal metastasis-related proteins, and showed synergistic action with chemotherapeutic agents in inhibiting the growth and survival of thyroid cancer cells.

Well differentiated thyroid cancer (WDTC), including papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC), is the most frequent subtype, but radioactive iodine (radioactive iodine) is the most frequent subtype in about 2/3 of patients. iodine; RAI) treatment does not work well. Undifferentiated thyroid cancer (UDTC), including anaplastic thyroid cancer (ATC), exhibits very aggressive characteristics characterized by external infiltration and distant metastasis of the thyroid gland, and a very poor prognosis (1-3). RAI treatment-resistant WDTC and UDTC patients do not respond to existing treatments, so new treatments that improve treatment efficacy against cancer cells are being studied (1-3).

Evodiamine is a natural indole alkaloid isolated from Evodia rutaecarpa, also called Wu-Zhu-Yu, and is a multi-target compound with a wide spectrum of biological activity (4). Evodiamine has traditionally been used to treat abdominal pain, vomiting, diarrhea, headache and postpartum bleeding (4). Evodiamine has beneficial activity in body temperature regulation, nociception, inflammation, obesity, cardiovascular disease, infectious diseases and Alzheimer's disease, and exerts anticancer effects in various cancer cells (4, 5). The mechanism of anticancer action of evodiamine is cell cycle arrest in the G2/M phase through activation of the Cdc2/cyclin B complex, survival-related proteins such as p21 and p53, Bcl2 family proteins, as well as multiplex including PI3K/AKT and NF-κB. It has been proposed to induce apoptosis by regulating the signaling pathway (6-15). However, the effect of evodiamine on the survival of thyroid cancer cells was not known.

Evodiamine prevents cancer progression by weakening the migration, invasion and metastasis of cancer cells (5, 6). Evodiamine inhibits cell migration and invasion of MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) in prostate and nasopharyngeal cancer cells and inhibits metastatic lesion formation in an animal model of colon cancer (17). -19). Regarding the epithelial mesenchymal transition (EMT), evodiamine inhibits the invasiveness of cancer cells activated by hepatocyte growth factor (HGF), and promotes self-renewal through the alleviation of EMT in gastric cancer stem cells. Eliminated and improved EMT activated by TGF-β in in vitro and in vivo models (15, 20-22). On the other hand, evodiamine exhibits cytotoxicity in breast cancer that does not work well with existing chemotherapy drugs (23, 24). In addition, evodiamine exhibits cell activity inhibitory properties and cytotoxicity in paclitaxel-resistant ovarian cancer cells (25). However, the combination effect of evodiamine and chemotherapeutic agents on thyroid cancer cells has not been identified.

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It is an object of the present invention to evaluate the effect of evodiamine on cell survival, proliferation, migration, and epithelial-mesenchymal metastasis, and to analyze the effect of evodiamine combined with a chemotherapeutic agent on thyroid cancer cell growth and survival to provide a more effective thyroid cancer therapeutic agent To provide.

The present invention proved for the first time that evodiamine causes apoptosis with co-mutation of survival-related proteins, and that inactivation of AKT enhances the negative effect of evodiamine on the survival of thyroid cancer cells. In addition, evodiamine showed a synergistic effect with chemotherapeutic agents in regulating EMT-related proteins to attenuate proliferation, migration, and EMT, and inhibit the growth and survival of thyroid cancer cells.

Evodiamine exhibits excellent antimitogenic action against hormone-sensitive prostate and breast cancer at the cellular level (5). As one of the proposed mechanisms, it has been reported that evodiamine blocks cell cycle progression at the G ₂ /M stage with stimulation of the Cdc2/cyclin B complex (6, 7). On the other hand, Bcl2 family proteins perform various major functions in cell homeostasis such as survival and proliferation (26). In this regard, the relative expression levels of the pro-viable protein Bcl2 and anti-viable protein Bax, called the Bcl2/Bax switch, affect the survival of cancer cells (27). Regarding the effect of evodiamine on survival-related proteins, including Bcl2 family proteins, evodiamine inhibits Bcl2, Bcl-xL, survivin, apoptotic protein inhibitor, MMP-9, cyclin D1 and cyclooxygenase 2 in various cancers. Induces cell death through (8). In addition, it was found that evodiamine induces apoptosis in A375-S2 melanoma cells through the ubiquitin-proteasome system, Bcl2/Bax ratio, and Bcl2 regulated by p21 and p53 (9, 10). In HepG2 liver cancer cells, evodiamine causes apoptosis by reducing the Bcl2/Bax ratio (11). Although evodiamine was reported to inhibit the survival and proliferation of ARO cells, which were considered ATC cells, these cells were identified as colon cancer cells (7, 28). Since TPC-1 and SW1736 cells used in this study were certified as PTC cells and ATC cells, respectively, this study is the first to report that evodiamine induces the death of true thyroid cancer cells (28). In summary, evodiamine decreased cell viability in thyroid cancer cells and dose-dependently increased the cytotoxic activity and percentage of apoptotic cells. Consequently, evodiamine decreases the Bcl2 protein level and does not change the Bax protein level, thereby reducing the Bcl2/Bax ratio. In addition, evodiamine increased the level of γH2AX and truncated PARP protein and decreased the level of survivin protein in thyroid cancer cells. These results indicate that evodiamine induces cell death by simultaneously changing the expression of Bcl2, survivin, and γH2AX in thyroid cancer cells. In addition, these results suggest that evodiamine exhibits cytotoxicity through the regulation of survival-related proteins in thyroid cancer cells.

PI3K/AKT signaling regulates many intracellular processes essential for the survival of healthy cells (29). The abnormality of PI3K/AKT signaling in thyroid cancer cells is involved in angiogenesis, and PI3K/AKT signaling was found to play an important role in cell survival in our previous studies (30-37). Regarding PI3K/AKT signaling involved in the anti-tumor action induced by evodiamine, evodiamine is through direct or indirect negative regulation of PI3K/AKT signaling in in vitro and in vivo pancreatic cancer models. It has been shown to improve the therapeutic efficacy of gemcitabine (12). In addition, evidence was presented that evodiamine induces apoptosis by reducing the level of phosphorylated-AKT protein in HepG2 liver cancer cells, and inhibits cell proliferation through PI3K/AKT signaling and inactivation of PTEN in osteosarcoma cells (11, 13). In A375-S2 melanoma cells, evodiamine induces apoptosis through inactivation of PI3K/protein kinase C and ERK cascade along with decrease of PI3K/AKT signal and increase of Fas-L/NF-κB signal and SIRT1 inactivation. (9, 10). In addition to PI3K/AKT signaling, evodiamine exhibits anticancer activity by regulating various signaling pathways such as NF-κB, mitogen activated protein kinase, and Wnt/β-catenin (8, 14, 15).

In this study, evodiamine increased the protein levels of phosphorylated-JNK, and did not affect the protein levels of total AKT, total NF-κB, total JNK and total and phosphorylated-ERK1/2, and phosphorylated-AKT and phosphorylated-NF- Reduced the protein level of κB. However, in cells treated with evodiamine, the PI3K inhibitor wotmannin, unlike inhibitors of NF-κB, JNK, and ERK1/2, decreases cell viability and increases cytotoxicity. In addition, wortmannin increased the level of cleaved PARP protein, did not affect the protein levels of total AKT and Bax, and decreased the protein levels of phosphorylated-AKT and Bcl2. These results indicate that inhibition of AKT in thyroid cancer cells stimulates evodiamine-induced cell death. Considering our previous studies that inhibition of AKT increases the cytotoxicity of various drugs in thyroid cancer cells, the results of this study mean that inhibition of AKT in thyroid cancer cells enhances evodiamine-induced cytotoxicity.

p21, p53 and matrix metalloproteinase (MMP) are associated with proliferation, migration, invasion and metastasis of cancer cells (41-43). In this regard, the present inventors have reported that p21, p53, MMP-2, and MMP-9 are involved in the proliferation and migration of thyroid cancer cells in conventional papers (37, 44). In addition to cell proliferation and cytotoxic effects, evodiamine inhibits cancer progression by interfering with the migration, invasion and metastasis of cancer cells (5, 16). In vitro and in vivo studies have shown that evodiamine inhibits cell migration and invasion through MMP-2 and MMP-9 in prostate and nasopharyngeal cancer cells, and reduces metastatic foci in nude mice challenged with colon cancer cells (17- 19). Regarding EMT, evodiamine has been reported to inhibit HGF-stimulated invasiveness in melanoma, lung cancer and colon cancer cells by inactivating bioactive HGF (20). In addition, evodiamine improves EMT in the process of standard Wnt/β-catenin signaling in gastric cancer stem cells, thereby reducing self-renewal and exhibiting inhibitory properties against TGF-β-stimulated EMT in NRK52E cells and rat liver fibrosis (15, 21, 22). In the present invention, evodiamine reduced cell growth and migration by reducing MMP-2 and MMP-9 as well as increasing p21 and p53 protein levels. TGF-β promoted cell growth and migration and induced a fibroblast-like phenotype, whereas evodiamine eliminated changes in cell growth, morphology and migration induced by TGF-β. In TGF-β-treated cells, protein levels of N-cadherin, phosphorylated-AKT, and MMP-2 were increased, and protein levels of p21 and p53 were decreased, but β-catenin, vimentin, slug, total AKT, and MMP-9. There was no change in protein level. Compared to the group administered with TGF-β alone, when evodiamine and TGF-β were administered together, the protein levels of p21 and p53 were increased and β-catenin, N-cadherin, vimentin, phosphorylated-AKT, MMP-2 and MMP The protein level of -9 decreased, while the slug and total AKT did not change. Taken together, evodiamine is simultaneously overexpressed in p21 and p53 in TGF-β-treated thyroid cancer cells, as well as simultaneous underexpression of β-catenin, N-cadherin, vimentin, phosphorylated-AKT, MMP-2 and MMP-9. To alleviate cell proliferation and migration. In addition, these results suggest that evodiamine negatively affects TGF-β-induced EMT in thyroid cancer cells through the regulation of EMT-related proteins.

According to treatment guidelines, chemotherapy may be considered for patients with tyrosine kinase inhibitor resistant WDTC and ATC (2, 3). In the past, doxorubicin has been approved as a monotherapy, but the clinical effect and response are not satisfactory in ATC patients (45). In addition, it has been reported that doxorubicin cannot remove cancer stem cells derived from ATC cells (46). Regarding other chemotherapeutic agents, paclitaxel was a relatively effective chemotherapeutic agent than doxorubicin traditionally used in ATC patients, but 17-allylamino-17-demethoxygeldanamycin, a heat shock protein 90 inhibitor. The present inventors have shown that induction of ATC cell death, the present inventors have an opposite effect on paclitaxel (32, 47). In addition, cisplatin alone or in combination with docetaxel showed an effect in ATC patients (3, 48). From the perspective of the combination of evodiamine and chemotherapeutic agents, evodiamine induces apoptosis in chemotherapeutic-resistant breast cancer in in vitro and in vivo models (23, 24). In addition, evodiamine inhibits cell proliferation and induces cytotoxic effects in paclitaxel-resistant A2780 ovarian cancer cells (25). In the present invention, when evodiamine was used in combination with the chemotherapeutic agents doxorubicin, paclitaxel, or cisplatin, all CI values were lower than 1.0 in terms of inhibition and mortality. These results show that evodiamine has a synergistic effect in inhibiting the growth and survival of thyroid cancer cells with doxorubicin, paclitaxel, or cisplatin. In addition, these results indicate that evodiamine synergizes with chemotherapeutic agents to inhibit thyroid cancer cell proliferation and induce cytotoxicity. Therefore, combination therapy of evodiamine and chemotherapeutic agents may be an attractive treatment for thyroid cancer.

In conclusion, the results of this study suggest that evodiamine exhibits anticancer activity by regulating survival-related proteins in thyroid cancer cells, and that inhibition of AKT increases evodiamine-induced cytotoxicity. In addition, evodiamine acts synergistically with chemotherapeutic agents in inhibiting cell proliferation and inducing cytotoxicity while inhibiting proliferation, migration and EMT by regulating EMT-related proteins in thyroid cancer cells. The present invention suggests that the combined use of evodiamine and chemotherapeutic agents is a promising treatment for thyroid cancer patients who do not respond to conventional treatments.

The present invention relates to a composition for treating thyroid cancer comprising evodiamine.

In addition, the present invention relates to a composition for treating thyroid cancer comprising evodiamine and a PI3K inhibitor.

In addition, the present invention relates to a composition for treating thyroid cancer, wherein the thyroid cancer is highly differentiated thyroid cancer and undifferentiated thyroid cancer.

In addition, the present invention relates to a composition for treating thyroid cancer, wherein the PI3K inhibitor is wortmannin.

In addition, the present invention relates to a composition for treating thyroid cancer comprising evodiamine and a chemotherapeutic agent showing an effect on thyroid cancer.

In addition, the present invention relates to a composition for treating thyroid cancer, wherein the thyroid cancer is a thyroid cancer resistant to conventional chemotherapeutic agents or conventional treatment methods.

In addition, the present invention relates to a composition for treating thyroid cancer, wherein the thyroid cancer is a thyroid cancer resistant to treatment with radioactive iodine.

In addition, the present invention relates to a therapeutic composition for thyroid cancer that is resistant to conventional chemotherapeutic agents or conventional treatment methods, including evodiamine and paclitaxel.

In addition, the present invention relates to a therapeutic composition for thyroid cancer that is resistant to conventional chemotherapeutic agents or conventional treatment methods, including evodiamine and cisplatin.

In addition, the present invention relates to a therapeutic composition for thyroid cancer that is resistant to conventional chemotherapeutic agents or conventional treatment methods, comprising evodiamine and doxorubicin.

The composition for treating undifferentiated thyroid cancer containing evodiamine alone or evodiamine and a chemotherapeutic agent of the present invention as an active ingredient can be formulated into an oral or injection form by a conventional method by combining it with a carrier commonly accepted in the pharmaceutical field. have. Oral compositions include, for example, tablets and gelatin capsules, in addition to the active ingredient, diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc). , Stearic acid and its magnesium or calcium salt and/or polyethylene glycol), and the tablets also contain binders (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone). ), and in some cases, a disintegrant (eg starch, agar, alginic acid or sodium salt thereof) or a boiling mixture and/or an absorbent, colorant, flavoring and sweetening agent. The composition for injection is preferably an isotonic aqueous solution or suspension, and the compositions mentioned are sterilized and/or contain adjuvants (e.g., preservatives, stabilizers, wetting or emulsifying agent solution accelerators, salts/or buffers for controlling the osmotic pressure). In addition, they may contain other therapeutically useful substances.

The pharmaceutical preparation prepared as described above may be administered orally, or parenterally, ie, intravenously, subcutaneously, or intraperitoneally as desired. The dose can be administered by dividing the daily dose of 0.0001 to 100 mg/kg in one to several times. The dosage level for a specific patient may vary depending on the patient's weight, age, sex, health status, administration time, administration method, excretion rate, and severity of disease.

According to the present invention, evodiamine is considered to be useful as a thyroid cancer therapeutic agent by exerting cytotoxicity in thyroid cancer cells through regulation of survival-related proteins.

In addition, according to the present invention, when a PI3K inhibitor such as wortmannin is added during evodiamine treatment, the inhibition of AKT enhances evodiamine-induced cytotoxicity, so that the combined use of evodiamine and PI3K inhibitor will be useful as a thyroid cancer treatment. do.

In addition, according to the present invention, when evodiamine is treated with chemotherapeutic agents such as doxorubicin, paclitaxel, and cisplatin, it has been shown to have a synergistic effect to inhibit the growth and survival of thyroid cancer cells. It is believed to be useful as

1 shows the effect of evodiamine on the survival of thyroid cancer cells. TPC-1 cells and SW1736 cells were treated with 1, 3, 5, 10 μM evodiamine for 48 hours, and then cell viability (A) and cytotoxic activity (B) were measured by CCK-8 assay and cytotoxicity assay, respectively. . C: TPC-1 cells and SW1736 cells were treated with 5 μM evodiamine for 48 hours, and then the percentage of apoptotic cells was measured using FACS analysis. D: TPC-1 cells and SW1736 cells were treated with 1, 3, 5, 10 μM evodiamine for 48 hours, and then the percentage of apoptotic cells was measured. E: TPC-1 cells and SW1736 cells were treated with 5 μM evodiamine for 48 hours, and protein levels of Bcl2, Bax, survivin, γH2AX and cleaved PARP were measured. All experiments were performed three times. The blot represents three independent experiments. Data are expressed as mean ± standard deviation. * p <0.05 is the value compared to the corresponding control, respectively.
Figure 2 shows the effect of evodiamine on the signaling protein in thyroid cancer cells. TPC-1 cells and SW1736 cells were treated with 5 μM evodiamine for 24, 48 hours, or 1, 2, 5 μM for 48 hours, and AKT and NF-kB (A) as well as JNK and ERK1/2 ( The total protein level and phosphorylated protein level of B) were measured. TPC-1 cells and SW1736 cells were treated with 2 μM of PI3K inhibitor wortmannin, 0.2 μM of NF-κB inhibitor BAY11-7082, 2 μM of JNK inhibitor SP600125 or 2 μM of MEK inhibitor PD98059, followed by treatment with 5 μM evodiamine for 48 hours. And, cell viability (C) and cytotoxicity (D) were measured. E and F: TPC-1 cells and SW1736 cells were treated with 2 μM wortmannin followed by 5 μM evodiamine for 48 hours. Protein levels of total and phosphorylated AKT proteins, Bcl2, Bax and truncated PARP were measured, quantified by densitometry and corrected with β-actin levels. All experiments were performed three times. The blot represents three independent experiments. Data are expressed as mean ± standard deviation. *p <0.05 is compared to the corresponding control, respectively; †P <0.05 compared to cells treated with evodiamine and DMSO; ‡P <0.05 compared to cells treated with evodiamine alone.
3 shows the effect of evodiamine on the proliferation and migration of thyroid cancer cells. A: TPC-1 cells and SW1736 cells were treated with 5 μM evodiamine for 48 hours, and the number of cells was counted to analyze cell growth. B and C: TPC-1 cells and SW1736 cells were treated with 5 μM evodiamine for 48 hours, and then cell migration was measured by a wound healing assay. D and E: TPC-1 cells and SW1736 cells were treated with 5 μM evodiamine for 48 hours. Protein levels of p21, p53, MMP-2 and MMP-9 were measured, quantified with a densitometer, and corrected with β-actin levels. All experiments were performed three times. The blot represents three independent experiments. Data are expressed as mean ± standard deviation. *p <0.05 is compared with the corresponding control, respectively.
Figure 4 shows the effect of evodiamine on TGF-β-stimulated epithelial-mesenchymal transition in thyroid cancer cells. TPC-1 cells and SW1736 cells were treated with 5 μM evodiamine and 100 ng/ml TGF-β for 48 hours, and cell growth (A), morphology (B) and migration (C) were measured. D and E: TPC-1 cells and SW1736 cells were treated with 5 μM evodiamine and 100 ng/ml TGF-β for 48 hours, followed by β-catenin, N-cadherin, vimentin, and slug total protein levels, total AKT Protein levels and phosphorylated AKT protein levels were measured. F and G: TPC-1 cells and SW1736 cells were treated with 5 μM evodiamine and 100 ng/ml TGF-β for 48 hours, and then protein levels of p21, p53, MMP-2 and MMP-9 were measured. All experiments were performed three times. The blot represents three independent experiments. Data are expressed as mean ± standard deviation. *p <0.05 compared to the corresponding control; †P <0.05 compared to cells treated with evodiamine and TGF-β. β-Cat: β-catenin; N-Cad: N-cadherin.
5 shows the effect of the combination of chemotherapeutic agents and evodiamine on the proliferation and survival of thyroid cancer cells. TPC-1 and SW1736 cells with 0.5, 1, 1.5, 2 μM evodiamine and 0.5, 1, 1.5, 2 μM doxorubicin, 0.5, 1, 1.5, 2 nM paclitaxel or 50, 100, 150, 200 μM cisplatin for 24 hours Processed. A and B: Cell growth was measured by counting the number of cells, and the inhibition rate was calculated as 100 − cell growth rate (%). C and D: Cell viability was measured using the CCK-8 assay, and mortality was calculated as 100 − cell viability (%). Combination index (CI) and isobologram were evaluated. All experiments were performed three times. Data are expressed as mean ± standard deviation.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those of ordinary skill in the art that the scope of the present invention is not limited only to the description of the examples.

reagent

Streptomycin/penicillin was purchased from Life Technologies (Carlsbad, CA, USA). Evodiamine, doxorubicin, paclitaxel, and cisplatin were purchased from BioVision (Linda, CA, USA) and then dissolved in dimethylsulfoxide (DMSO). Bcl2, Bax, survivin, phosphorylated-histone H2A.X (γH2AX), truncated poly (ADPribose) polymerase (PARP), total and phosphorylated NF-κB (Ser536), total and phosphorylated JNK (C-Jun N- terminal kinase) (Thr183/Tyr185), total and phosphorylated ERK (extracellular signal-regulated kinase) 1/2 (Thr402/Tyr404), p21, p53, MMP-2, MMP-9, β-catenin, N- Caderin, vimentin, and slug were purchased from Cell Signaling Biotechnology (Danvers, MA, USA). The primary antibody against total and phosphorylated-AKT (Ser473) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the primary antibody against β-actin was purchased from Sigma (St. Louis, MO, USA). I did. All other reagents were purchased from Sigma unless otherwise specified.

For the experiment, TPC-1 human thyroid papillary cancer cells were obtained from Professor Young-Joo Park (Department of Endocrinology and Metabolism, Seoul National University College of Medicine), and cultured in RPMI1640 supplemented with 10% heat-treated inactivated FBS and 1% streptomycin/penicillin. SW1736 human anaplastic thyroid cancer cells were purchased from Cell Lines Service (CLS GmbH, Eppelheim, Germany) and cultured in RPMI1640 to which 2 mM L-glutamine, 10% heat-treated inactivated FBS and 1% streptomycin/penicillin were added. Cells were supplemented with fresh medium at regular time intervals. Treatment and experiments were carried out in a state of 70% floor area ratio.

CCK -8 analysis

Cell viability was measured with a CCK-8 assay kit (Dojindo laboratories, Kumamoto, Japan). Cells (5 x 10 ³ /100 μl) in each well of a 96-well plate were incubated overnight, and then treated with an agent for an additional 4 hours at 37°C. Absorbance was measured using Glomax™ Discover System GM3000 (Promega, Madison, WI, USA). All experiments were performed three times.

Cytotoxicity assay

Cytotoxicity was measured by LDH cytotoxicity assay kit (BioVision, Linda, CA, USA). After culturing the cells ^{(5 × 10 3/100 ㎕} ) in each well of a 96-well plate and centrifuged for 10 min at 250 g. 100 μl of the supernatant was transferred to a clear 96-well plate. After adding the reaction mixture (2.5 μl catalyst solution in 112.5 μl dye solution), the cells were incubated at room temperature for 30 minutes. Absorbance was measured using Glomax™ Discover System GM3000 (Promega). All experiments were performed three times.

FACS analysis

Apoptosis cells were analyzed with Annexin V-FITC Apoptosis Detection Kit (BD Biosciences Pharminogen, San Diego, CA, USA). Cells (1 × 10 ⁵ /ml) in each well of a 6-well plate were cultured, harvested, washed, and then fixed according to the manufacturer's protocol. 1 × binding buffer containing FITC annexin V and/or propidium iodide (PI) was added at room temperature for 15 minutes, followed by CytoFLEX™ Flow Cytometer (Beckman Coulter Inc., Brea, CA, USA) And CytExpert software (Beckman Coulter Inc., Brea, CA, USA). All experiments were performed three times.

Cell count

Cell growth was measured by cell number using trypan blue. Cells (1 × 10 ⁵ /500 μl) in each well of a 12-well plate were cultured and then mixed with trypan blue dye at 37°C. Stained cells were counted using a hemocytometer. All experiments were performed three times.

Wound healing analysis

Cell migration was measured with a CytoSelect™ 24-well wound treatment assay kit (Cell Biolabs, San Diego, CA, USA). After creating a wound area (0.9 mm), it was incubated at 37°C. After confirming the wound closure with an optical microscope, the cell migration rate was calculated according to the following formula: Cell migration = [cell migration length (nm)/migration time (hr)]. All experiments were performed three times.

Western Blotting

Total protein was extracted with RIPA buffer (Sigma) containing 1 × protease inhibitor cocktail and 1 × phosphatase inhibitor cocktail set V (Calbiochem, La Jolla, CA, USA). Western blotting was performed using a specific primary antibody and peroxidase-binding anti-rabbit or anti-mouse secondary antibody. Bands were detected using the ECL Plus Western Blotting Detection System (Thermo Fisher Scientific, Rockford, IL, USA). Protein levels were quantified using ImageJ software (NIH) and corrected for β-actin protein levels. The relative level of the protein to the β-actin protein level was determined. All experiments were performed three times.

Drug combination analysis

Combination index (CI) and isobologram were calculated using CalcuSyn program version 2.11 (Biosoft, Great Shelford, Cambridge, UK), and the effect of drug interaction was quantitatively analyzed. CI values less than 1.0, 1.0 and greater than 1.0 represent synergistic, additive and antagonistic effects, respectively. The isobologram plots the dose of each drug required for 50% inhibition (ED ₅₀ ) on the x and y axis and connects the dots to draw the ED ₅₀ isobologram line. Combination data points above, below, and above the line segment represent additive, synergistic and antagonistic effects, respectively. All experiments were performed three times.

Statistical analysis

All data were expressed as mean ± standard deviation (S.E). Data were appropriately analyzed by unpaired Student's t-test or ANOVA. A p value of less than 0.05 was considered to be indicative of statistical significance. All analyzes were performed using SPSS program version 24.0 (SPSS, Chicago, IL, USA).

Outcome 1: Evodiamine is Induces the death of thyroid cancer cells.

To evaluate the effect of evodiamine on cell survival, TPC-1 and SW1736 cells were treated with 1, 3, 5, and 10 μM evodiamine for 48 hours, and then the cell viability (FIG. 1a) and cytotoxicity (FIG. 1b) were CCK. Measured by -8 assay and cytotoxicity assay, respectively. After treatment, cell viability decreased and cytotoxic activity increased in a dose dependent manner. When the cells were treated with 5 μM evodiamine for 48 hours, the percentage of apoptotic cells measured using FACS analysis increased (FIG. 1C ). In addition, treatment with 1, 3, 5 and 10 μM evodiamine for 48 hours increased the proportion of apoptotic cells in a dose-dependent manner (FIG. 1D ). To evaluate the effect of evodiamine on survival-related protein expression, extracts of cells treated with 5 μM evodiamine for 48 hours were Western blot using antibodies against Bcl2, Bax, survivin, γH2AX and cleaved PARP. It was examined as (Fig. 1e). After treatment, γH2AX and truncated PARP protein levels were increased, and Bcl2 and survivin protein levels decreased without changes in Bax protein levels.

Outcome 2: AKT Inactivation is With evodiamine Enhances the induced death of thyroid cancer cells.

To measure the effect of evodiamine on the signal protein, extracts of cells treated with 5 μM evodiamine for 24, 48 hours or 1, 2 and 5 μM evodiamine for 48 hours were total and phosphorylated AKT, NF-kB, It was examined by western blot using antibodies against JNK and ERK1/2 (Figs. 2A and 2B). After treatment, the protein levels of phosphorylated-JNK increased, and the protein levels of phosphorylated-AKT and phosphorylated-NF-κB decreased. In contrast, protein levels of total AKT, total NF-κB, total JNK, and total and phosphorylated ERK1/2 did not change.

To demonstrate the role of signaling proteins in the survival of cells exposed to 5 μM evodiamine for 48 hours, cells were treated with the PI3K inhibitor wortmannin, the NF-κB inhibitor BAY11-7082, the JNK inhibitor SP600125, or the MEK inhibitor PD98059. It was pretreated. After treatment, cell viability (Fig. 2c) and cytotoxic activity (Fig. 2d) were measured. Wortmannin decreased cell viability and increased cytotoxicity, but BAY11-7082, SP600125 and PD98059 did not. When cells were incubated with wortmannin before treatment with 5 μM evodiamine for 48 hours, the protein levels of cleaved PARP increased and the protein levels of phosphorylated-AKT and Bcl2 decreased. Compared with cells treated with evodiamine alone, total AKT and Bax protein levels in cells treated with wortmannin and evodiamine were not affected (FIGS. 2E and 2F ).

Outcome 3: Evodiamine is Inhibits the proliferation and migration of thyroid cancer cells.

In order to confirm the effect of evodiamine on cell proliferation, cells were treated with 5 μM evodiamine for 48 hours, and then cells were counted to evaluate cell growth (FIG. 3A). Cell growth decreased after evodiamine treatment. In order to investigate the effect of evodiamine on cell migration, cells were treated with 5 μM evodiamine for 48 hours and then evaluated by a wound healing assay (Figs. 3b and 3c), and proteins of p21, p53, MMP-2, and MMP-9 Levels were checked by Western blot (FIGS. 3D and 3E ). After treatment, the protein levels of p21 and p53 increased, while the cell migration and protein levels of MMP-2 and MMP-9 decreased.

Outcome 4: Evodiamine is In thyroid cancer cells TGF -β activation EMT Weakens.

Evodiamine slows cancer progression by inhibiting the migration, invasion and metastasis of cancer cells (5, 16). In this aspect, evodiamine inhibits cancer cell invasion and metastasis through the regulation of MMP-2 and MMP-9 in prostate and nasopharyngeal cancer cells, and inhibits the formation of metastatic lesions in a mouse model of colon cancer (17-19). Regarding EMT, evodiamine attenuates HGF-induced invasiveness in melanoma, lung cancer and colon cancer cells and prevents self-renewal through EMT inhibition in gastric cancer stem cells (15, 20). In addition, evodiamine inhibits TGF-β-induced EMT in rat NRK52E cells and liver fibrosis (21, 22). The present inventors investigated whether evodiamine affects TGF-β-induced EMT in thyroid cancer cells.

To investigate the effect of evodiamine on EMT induced by TGF-β, cells were treated with 5 μM evodiamine and 100 ng/ml TGF-β for 48 hours, followed by cell growth (Fig. 4a), morphology (Fig. 4b). And movement (Figure 4c) was measured. After treatment, TGF-β increased cell growth and migration, and induced a fibroblast-like phenotype. Evodiamine attenuated the changes induced by TGF-β in cell growth, morphology and migration.

When treated with 100 ng/ml TGF-β for 48 hours, protein levels of N-cadherin, phosphorylated-AKT and MMP-2 were increased, and proteins of β-catenin, vimentin, slug, total AKT and MMP-9 The protein levels of p21 and p53 were decreased without affecting the level (Fig. 4d-g). Compared to cells treated with only TGF-β, β-catenin, N-cadherin, vimentin, phosphorylation- without affecting the protein levels of slug and total AKT in cells treated with evodiamine and TGF-β together. It decreased the protein levels of AKT, MMP-2 and MMP-9.

Outcome 5: Evodiamine is It acts synergistically with chemotherapeutic agents when it shows cytotoxic and cytotoxic effects on thyroid cancer cells.

To evaluate the cell proliferation inhibitory effect of the combination of evodiamine and chemotherapeutic agent, the interaction was analyzed by treating the cells with evodiamine and doxorubicin, paclitaxel, or cisplatin, and then obtaining CI using the Chou-Talalay equation. CI <1.0 represents a synergistic effect, CI = 1.0 represents additive, and CI> 1.0 represents an antagonistic effect (FIGS. 5A and 5B, Table 1). Cell growth was analyzed by counting cells, and the inhibition rate was calculated as 100 − cell growth rate (%).

Upon combination treatment, all CI values were lower than 1.0, and all points of the combination data were located below the isobologram line at ED ₅₀ . This suggests a synergistic action between evodiamine and chemotherapeutic agents in inducing cell proliferation inhibitory activity in thyroid cancer cells.

Next, cells were treated with evodiamine and the chemotherapeutic agents in order to analyze the cytotoxic effect of the combination of evodiamine and chemotherapeutic agent (FIGS. 5c and 5d, Table 2). Cell viability was measured by the CCK-8 assay, and the death rate was calculated as 100 − cell viability (%). After combination treatment, all CI values were less than 1.0, and the points of the combination data were all located below the isobologram line in ED ₅₀ . This suggests that evodiamine has a synergistic effect on thyroid cancer cytotoxicity with chemotherapy.

Claims (10)

Evodiamine; And
A composition for treating thyroid cancer comprising wortmannin as a PI3K inhibitor.
The method according to claim 1,
The thyroid cancer treatment composition for thyroid cancer, characterized in that the thyroid cancer is highly differentiated thyroid cancer and undifferentiated thyroid cancer.
A composition for treating thyroid cancer comprising evodiamine and paclitaxel.
A composition for treating thyroid cancer comprising evodiamine and cisplatin.
A composition for treating thyroid cancer comprising evodiamine and doxorubicin.
The method according to any one of claims 3 to 5,
The thyroid cancer treatment composition, characterized in that the thyroid cancer is resistant to conventional chemotherapy or conventional treatment methods.
The method according to any one of claims 3 to 5,
The thyroid cancer treatment composition, characterized in that the thyroid cancer is a thyroid cancer resistant to radioactive iodine treatment.

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