Pharmaceutical composition comprising NSC-38270 for inhibiting metastasis hepatocellular carcinoma

Abstract

The present invention relates to a composition comprising NSC-38270 or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition of the present invention can prevent and / or treat metastasis of liver cancer by effectively inhibiting the metastasis and invasion of liver cancer cells through the inhibition of the major EMT-TF gene Twist1 and the induction of the E-cadherin1 (CDH1) gene.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for preventing and inhibiting metastasis of liver cancer cells, comprising a compound (NSC-38270) represented by Chemical Formula 1 as an active ingredient.

[0002] The present invention relates to a health functional food composition for preventing and inhibiting metastasis of liver cancer cells, comprising a compound (NSC-38270) represented by Chemical Formula 1 as an active ingredient.

[0003] Background Technology

[0004] Liver cancer refers to a malignant tumor originating from liver cells, which make up the majority of the liver. In a broad sense, it includes all types of malignant tumors that occur in the liver (e.g., intrahepatic cholangiocarcinoma) and even metastatic liver cancer resulting from the spread of cancer from other organs; however, because hepatocellular carcinoma (HCC) is the most common type of liver cancer, the term generally refers only to hepatocellular carcinoma arising from liver cells.

[0005] Hepatocellular carcinoma (HCC) is the most frequently occurring malignant disease in patients with chronic liver disease, and it is the third most common tumor in the world, claiming 500,000 lives annually, with a high incidence of metastasis via the portal vein. Although various treatment techniques have been introduced into clinical practice, the survival rate of HCC patients remains poor. In Korea, the incidence of HCC has not improved over the past 20 years and is nearly identical to the mortality rate. Chronic hepatitis caused by infection with the hepatitis B or C virus, as well as exposure to carcinogens such as alcohol and aflatoxin B1, are known major risk factors for HCC. In particular, HCC is characterized by its aggressive nature and high potential for metastasis; it spreads rapidly to other parts of the body, making treatment difficult and resulting in a poor prognosis.

[0006] Epithelial-mesenchymal transition (EMT) is an essential process in various developmental stages, such as mesoderm formation and neural tube formation, but it is also known to be closely associated with organ fibrosis and the initiation of cancer progression and metastasis. In particular, EMT-TFs (EMT-related transcription factors), including the Snail, Twist, and Zeb families, primarily inhibit the expression of adhesion proteins called E-cadherins, which maintain intercellular junctions; among these, Twist1 is a key mediator of EMT and induces cancer metastasis by increasing the migration and invasion of cancer cells. As such, the metastatic process of liver cancer cells is also known to be associated with EMT, making further research necessary (Nagai, Tomoyuki, et al. Molecular cancer therapeutics 10.1 (2011): 169-177.).

[0007] NSC-38270 is the largest antibiotic-producing gene. Streptomyces It is produced by (Kampfer, Peter. The prokaryotes 3 (2006): 538-604.). Olivomycin A is an anticancer antibiotic used to treat testicular cancer, reticular sarcoma, and various tumors, but it has high cytotoxic effects. NSC-38270 is a structural isomer of olibomycin A (Cas No. 102647-16-5, CID 122806) and is known to exhibit anticancer effects (Tevyashova AN, Pharmaceutical Chemistry Journal 50.7 (2016): 425-430., Walling, JA, Synthetic approaches to aromatic antitumor agents and antibiotics (1986).).

[0008] As prior art, prior literature [Haller, Daniel G., et al. Cancer Treatment Reports 62.4 (1978): 563-565.] describes the antitumor effects of NSC-38270 in patients at the non-clinical and clinical stages, and prior literature [Gozari, Mohsen, et al. 3 Biotech 9.12 (2019): 439.] describes the cytotoxic activity of SP85, an olibomycin A analog, against HepG2, an HCC cell line.

[0009] However, nothing has been revealed regarding the cytotoxicity, anti-invasiveness, and particularly the effects of NSC-38270 on liver cancer metastasis in hepatocellular carcinoma.

[0010] Accordingly, the inventors of the present invention completed the present invention by confirming, through diligent research efforts to develop an effective therapeutic agent for liver cancer metastasis, that NSC-38270 inhibits liver cancer metastasis by specifically inhibiting the migration and invasion of liver cancer cells without affecting normal cells. The problem to be solved

[0011] The objective of the present invention is to provide a pharmaceutical composition and a health food composition for preventing and inhibiting metastasis of liver cancer cells, comprising NSC-38270 as an active ingredient. means of solving the problem

[0012] To achieve the above objective, the present invention provides a pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] In one embodiment of the present invention, in addition to the active ingredient, it may further include a pharmaceutically acceptable carrier, excipient, or diluent.

[0014] In another embodiment of the present invention, the active ingredient may be achieved by inhibiting the migration, invasion, and both of the liver cancer cells.

[0015] In another embodiment of the present invention, the active ingredient may be achieved by inhibiting epithelial-mesenchymal transition.

[0016] In another embodiment of the present invention, the active ingredient may inhibit the expression of the Twist1 gene, which is an EMT-TF gene.

[0017] In another embodiment of the present invention, the active ingredient may induce the expression of the E-cadherin1 (CDH1) gene.

[0018] In another embodiment of the present invention, the active ingredient may not affect the expression of the EMT-TF gene, such as Snail1 or Zeb1.

[0019] In another embodiment of the present invention, the pharmaceutical composition may further comprise an anticancer agent.

[0020] The present invention provides a health food composition for preventing and inhibiting liver cancer cell metastasis, comprising a compound represented by Chemical Formula 1 (NSC-38270) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0021] In addition, the present invention provides a screening method for a substance having activity to inhibit metastasis of liver cancer cells, comprising: 1) treating a liver cancer cell line with a compound represented by Formula 1 as a positive control and measuring the level of Twist1 mRNA or protein; 2) treating a liver cancer cell line with a candidate substance and measuring the level of Twist1 mRNA or protein; and 3) determining that the candidate substance has activity to inhibit metastasis of liver cancer cells if the measurement result of step 2) is lower than the measurement result of step 1). Effects of the invention

[0023] The compound represented by Formula 1 according to the present invention (NSC-38270) or a pharmaceutically acceptable salt thereof can be usefully utilized to prevent and inhibit the division and metastasis of liver cancer cells by inhibiting metastasis and invasion through the inhibition of the major EMT-TF gene Twist1 and the induction of the E-cadherin1 (CDH1) gene. Brief explanation of the drawing

[0024] Figure 1 shows the cell viability of Huh7 cells exposed to NSC-38270 for 24 hours. Figure 2 shows the results of a wound healing assay of cells treated with NSC-38270 for 24 hours. Figure 3 shows the results of transwell migration and invasion analysis of cells treated with NSC-38270. Figure 4 shows the protein expression of Twist1, a major EMT-TF factor, when NSC-38270 was treated to Huh7 cells at different concentrations or times. Figure 5 shows the protein expression of Twist1 when MG132 was pretreated before treatment with NSC-38270. Figure 6 shows the protein expression of other EMT-TF factors, Snail1 and Zeb1, when NSC-38270 was administered to Huh7 cells for 24 hours. Figure 7 shows the mRNA levels of Twist1 and CDH1 when Huh7 cells were treated with NSC-38270 for 24 hours. Figure 8 shows the cell growth of Huh7 treated with NSC-38270. Figure 9 shows the morphological analysis of Huh7 treated with NSC-38270. Figure 10 shows the apoptosis of Huh7 cells treated with NSC-38270 for 24 hours. Figure 11 shows the activation of histone H2A.X in Huh7 cells treated with NSC-38270 for 24 hours. Figure 12 shows the protein expression of cleaved caspase-3 and cleaved PARP in Huh7 cells treated with NSC-38270 for 24 hours. Figure 13 shows the mRNA levels of Bax or Bcl2 in Huh7 cells treated with NSC-38270 for 24 hours. Figure 14 shows cell growth in normal hepatocytes, such as THLE-2 cells or Chang hepatocytes, treated with NSC-38270. Figure 15 shows the morphological analysis of normal hepatocytes, THLE-2 cells or Chang hepatocytes, treated with NSC-38270. Figure 16 shows the analysis of apoptosis in normal hepatocytes, THLE-2 cells or Chang hepatocytes, treated with NSC-38270 for 24 hours. Figure 17 shows the activation of histone H2A.X in normal hepatocytes, THLE-2 cells or Chang hepatocytes, treated with NSC-38270 for 24 hours. Specific details for implementing the invention

[0025] The present invention will be described in detail below.

[0027] The present invention provides a pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis comprising NSC-38270 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0028] The present invention provides a health food composition for preventing and inhibiting liver cancer cell metastasis, comprising NSC-38270 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0029] NSC-38270 is a structural isomer of olivomycin A and is represented by [Chemical Formula 1].

[0030] [Chemical Formula 1]

[0031]

[0032] The pharmaceutical composition of the present invention may additionally include a carrier, excipient, or diluent as an active ingredient, in addition to NSC-38270 or a pharmaceutically acceptable salt thereof.

[0033] In the present invention, the term "metastasis" refers to the movement of cancer cells to other tissues or organs through the blood vessels of the primary cancer, and the present invention includes all cases in which hepatocellular carcinoma is metastasized to another organ or cancer cells are metastasized from another organ to cause hepatocellular carcinoma.

[0034] In the present invention, the terms “hepatocellular carcinoma” or “liver cancer” refer to malignant tumors originating from liver cells, which constitute the majority of the liver. In a broad sense, it includes all types of malignant tumors occurring in the liver (e.g., intrahepatic cholangiocarcinoma) and even metastatic hepatocellular carcinoma resulting from the metastasis of cancer from other organs to the liver. For example, in the present invention, “liver cancer” includes, but is not limited to, hepatocellular carcinoma, cholangiocarcinoma, and angiosarcoma, and most preferably refers to hepatocellular carcinoma.

[0035] In the present invention, the phrase 'prevention and inhibition of liver cancer metastasis' is achieved by inhibiting the division, migration, or invasion of liver cancer cells, particularly invasion into normal cells, but is not limited thereto.

[0036] In the present invention, "prevention" refers to any act of suppressing or delaying the symptoms of hepatocellular carcinoma by administering a composition containing NSC-38270 or a pharmaceutically acceptable salt thereof as an active ingredient according to the present invention, and in particular means delaying the metastasis of hepatocellular carcinoma as described above.

[0037] In the present invention, NSC-38270 or a pharmaceutically acceptable salt thereof exhibits activity that inhibits the process of epithelial-mesenchymal transition (EMT).

[0038] The term "epithelial-mesenchymal transition (EMT)" refers to the process in which epithelial cells lose cell polarity and cell-cell adhesion, acquire metastatic and invasive capabilities, and become mesenchymal stem cells, which are multipotent stromal cells capable of differentiating into various types of cells.

[0039] The aforementioned EMT is essential for many developmental processes, including mesoderm formation and neural tube formation. Furthermore, EMT is known to be involved in wound healing, tissue fibrosis, and the initiation of metastasis in cancer progression.

[0040] NSC-38270 or a pharmaceutically acceptable salt thereof may exhibit activity that inhibits the expression of the EMT-TF gene, Twist1.

[0041] NSC-38270 or a pharmaceutically acceptable salt thereof may exhibit activity that induces the expression of the E-cadherin1 (CDH1) gene.

[0042] NSC-38270 or its pharmaceutically acceptable salt may additionally contain an anticancer agent.

[0043] The above anticancer agent may include, without limitation, any anticancer agent known in the field that is used for the treatment of liver cancer or inhibition of metastasis, and may be one or more anticancer agents selected from the group consisting of oxaliplatin, pemetrexed, cisplatin, irinotecan, gemcitabine, carboplatin, bortezomib, fluorouracil (5-FU), cyclophosphamide, paclitaxel, vincristine, etoposide, and doxorubicin.

[0044] The pharmaceutical composition of the present invention can be administered to a subject in a pharmaceutically effective amount.

[0045] In the present invention, the term "pharmaceuticalally effective amount" refers to a reasonable amount applicable to medical treatment, meaning an amount sufficient to treat a disease. The criteria for this amount may be determined based on the patient's disease, severity, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitant components, and other factors. The pharmaceutical composition of the present invention may be administered in combination with an individual therapeutic agent or other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, the dosage may be determined to a level that minimizes side effects, and this can be easily determined by a person skilled in the art. Specifically, the dosage of the pharmaceutical composition may vary depending on the patient's age, weight, severity, gender, etc., and generally, an amount of 0.001 to 150 mg, more preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, 1 to 3 times a day. However, this is merely illustrative, and the dosage may be set differently as necessary.

[0046] The term "subject" as used in the present invention may refer to any animal, including humans, that has developed or is likely to develop hepatocellular carcinoma. Such animals may include not only humans but also mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar symptoms, but are not limited thereto.

[0047] The pharmaceutical composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. Pharmaceutically acceptable carriers may include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and a mixture of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, target organ-specific antibodies or other ligands may be bound to the carrier to act specifically on target organs. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the relevant technical field or by using the method disclosed in Remington's literature (Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton, PA).

[0048] The pharmaceutical composition of the present invention may be administered orally or parenterally (intravenous injection, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time, and may be selected in an appropriate form by those skilled in the art.

[0049] The term "health functional food" in the present invention refers to a food manufactured using raw materials or ingredients that have functions useful to the human body or nutrients that are easily deficient in daily meals, and which helps maintain human health; however, it is not limited thereto and is used to include all health foods in the conventional sense.

[0050] The form and type of the health functional food are not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids, and pills.

[0051] The present invention comprises the steps of: 1) treating a liver cancer cell line with NSC-38270 or a pharmaceutically acceptable salt thereof as a positive control and measuring the level of Twist1 mRNA or protein;

[0052] 2) A step of treating liver cancer cell lines with a candidate substance and measuring Twist1 mRNA or protein levels;

[0053] 3) A step of determining the candidate substance as having activity to inhibit liver cancer cell metastasis if the measurement result of Step 2) is lower than the measurement result of Step 1);

[0054] A screening method for substances having liver cancer cell metastasis inhibitory activity is provided, comprising

[0055] In the above method, the liver cancer cells of step 1) may be any one selected from the group consisting of Huh7, Hep3B, HepG2 and SNU878 cells.

[0056] In the above method, the method for measuring the mRNA or protein level in step 2) may be RT-qPCR or Western blot.

[0057] In the above method, after step 3), a step of determining whether a substance determined to have activity to inhibit metastasis of liver cancer cells increases the expression of the CDH1 gene may be further included.

[0058] In a specific embodiment of the present invention, the inventors confirmed the inhibitory activity of NSC-38270 on the motility and invasiveness of liver cancer cells (Figs. 2 and 3) through the inhibition of the EMT gene Twist1 (Figs. 4 and 7) or the induction of E-cadherin (Fig. 7), and confirmed that it promotes selective apoptosis of liver cancer cells (Figs. 8 to 13) without affecting normal liver cells (Figs. 14 to 17).

[0060] Example 1: Cytotoxicity Analysis of NSC-38270

[0061] To analyze the cytotoxicity of NSC-38270 in HCC and determine the optimal concentration, the viability of Huh7 cells treated with NSC-38270 was analyzed. Huh7 cells were seeded into 96-well plates and cultured overnight. Subsequently, once full confluency was reached, the cells were treated with NSC-38270 in medium containing 1% or 10% FBS. 24 hours after NSC-38270 treatment, cell viability was evaluated using EZ-Cytox reagent (DOGEN, Seoul, Korea) according to the manufacturer's instructions.

[0062] When Huh7 cells were treated with NSC-38270 in a medium containing 10% FBS for 24 hours, cell viability was maintained at over 80% at 50 nM, which was used as the maximum concentration of the experiment (Fig. 1).

[0064] Example 2: Wound healing assay of NSC-38270

[0065] To evaluate cell motility and invasiveness in Huh7 cells treated with NSC-38270, a wound healing assay was performed. Huh7 cells were seeded in 24-well plates at 90% confluence. Scratches were made in the cells using a scraper tip, and the detached cells were washed with PBS. Then, NSC-38270 was mixed into RPMI containing 1% FBS and the cells were treated for 24 hours. A positive control was treated in the same manner as NSC-38270, and 100 nM Cucurbitacin E (CuE), known to significantly inhibit cell proliferation and migration of Huh7 cells, was used as the positive control. The scratched areas were imaged using a JuLI™ Stage real-time cell history recorder (NanoEnTek, Inc.), and ImageJ software was used to quantify the scratch area and the area occupied by migrated cells, with the results displayed in a graph.

[0066] As a result, Huh7 cells treated with NSC-38270 delayed wound healing (Fig. 2).

[0068] Example 3: Analysis of Transwell Mobility and Invasiveness of NSC-38270

[0069] To evaluate cell motility and invasiveness in Huh7 cells treated with NSC-38270, a transwell migration and invasion assay was performed by treating with NSC-38270 for a relatively short period (<24 hours) to exclude cell growth effects.

[0070] Huh7 cells (5×10⁻⁶ 4Canine cells were seeded into the inner chamber of a Transwell plate (pore size, 8 μM, polycarbonate membrane; SPL Life Sciences, Korea) along with NSC-38270 in medium containing 1% FBS. Glaucarubinone (GCB, 1 μM), known to significantly inhibit cell migration and invasion, was used as the positive control for the Transwell assay. The outer wells were filled with medium containing 10% FBS, and the plates were incubated for 21 hours at 37°C in a 5% CO2 environment, after which the inner chamber was washed once with PBS. The chamber was then fixed with 4% paraformaldehyde in PBS and stained with 0.5% crystal violet. Cells attached to the inner membrane were removed using a cotton swab. Stained cells attached to the outer membrane were randomly observed and photographed. The area of ​​the migrated cells was measured using ImageJ software (NIH, Bethesda, MD, USA), and the invasion analysis was performed in the same manner, except for the step of coating the inner membrane of the inner chamber with diluted Matrigel (BD Biosciences, San Jose, CA, USA) before seeding the cells.

[0071] As a result, NSC-38270 inhibits the migration and invasion of Huh7 cells, which can potentially infiltrate other tissues and cause cancer metastasis (Fig. 3).

[0073] Example 4: Confirmation of EMT-TF related gene expression levels in Huh7 cells treated with NSC-38270

[0074] To evaluate the effect of NSC-38270 on major EMT-TF expression, protein and mRNA levels of major EMT-TF were evaluated in Huh7 cells treated with NSC-38270.

[0076] 4-1. Evaluation of Protein Levels of EMT-TF Related Genes in Huh7 Cells Treated with NSC-38270

[0077] To determine the protein expression level of Twist1, a major EMT-TF gene, NSC-38270-treated cells were washed once with PBS and then lysed in lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5% IGEPAL, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 mM sodium orthovanadate, and 10 mM NaF). Total cell lysate protein concentration was measured using the Bradford Protein Assay Kit (Bio-Rad, Hercules, CA, USA; 5000006). Protein samples of the same concentration were mixed with 5X sodium dodecyl sulfate (SDS) sample buffer (250 mM Tris-HCl, pH 6.8, 5% 2-mercaptoethanol, 10% SDS, 0.5% bromophenol blue, and 50% glycerol) and boiled at 100 °C for 5 minutes. The boiled lysate was separated on a 12% polyacrylamide gel by gel electrophoresis at 80 V for 2 hours. After electrophoresis, the protein bands were transferred to a nitrocellulose membrane (Bio-Rad; 1620112). The membrane was blocked with 5% skim milk for 1 hour and then incubated overnight at 4 °C with the appropriate antibody in 5% BSA. After removing the unbound primary antibody, the membrane was incubated at room temperature (RT) for 2 hours with a secondary antibody bound to HRP (horseradish peroxidase) contained in 5% skim milk. Immuno-reaction bands were visualized on Dyne ECL (Dyne Bio, Inc., Korea) using a cooled charge-coupled device camera system (AE-9150, ATTO Technology, Tokyo, Japan).

[0078] As a result, NSC-38270 downregulated the protein expression of Twist1 in a concentration-dependent manner, and in particular, the protein level of Twist1 decreased rapidly 3 hours after treatment with 50 nM NSC-38270 (Fig. 4).

[0079] Since the protein level of Twist1 decreased rapidly within a considerably short time after treatment with NSC-38270, in order to confirm the relationship between NSC-38270 and Twist1 degradation, the Twist1 protein level was evaluated by adding a pretreatment step with MG132, which inhibits proteasome-mediated protein degradation.

[0080] As a result, Twist1 protein levels decreased in a concentration-dependent manner with NSC-38270, similar to the case where MG132 was not treated (Fig. 5). This suggests that NSC-38270 does not induce Twist1 protein degradation.

[0081] In addition, the protein levels of Snail1 and Zeb1, major EMT-TFs other than Twist1 protein, were evaluated in Huh7 cells treated with NSC-38270. As a result, unlike Twist1, the protein levels of Snail1 and Zeb1 in Huh7 cells treated with NSC-38270 were not significantly altered (Fig. 6).

[0083] 4-2. Evaluation of mRNA levels of EMT-TF-related genes in Huh7 cells treated with NSC-38270

[0084] To determine the mRNA expression levels of Twist1, a major EMT-TF gene, and the E-cadherin gene (CDH1), one of the target genes of EMT-TF, cells were seeded into 12-well plates and treated with NSC-38270 for 24 hours. Total RNA was extracted using Labozol reagent (Cosmo Genetech, Korea), and 1 μg of RNA was reverse transcribed into complementary DNA (cDNA) using the TOPscript™ cDNA Synesis Kit (Enzynomics, Korea; EZ005M). Real-time PCR was performed using a CFX Connect real-time thermal cycler (Bio-Rad) in conjunction with Dyne qPCR 2X PreMIX (Dyne Bio, Inc.). cDNA was initially denatured at 95 °C for 5 minutes, followed by 94 °C for 5 seconds, and then amplified for 39 cycles at 55 to 61 °C for 30 seconds. The fluorescence values ​​of the PCR amplicon were normalized using the GAPDH gene, and 2 -△△Cq Depending on the method, the gene expression level was expressed as a ratio with the control group set to 100%. The list of primers is listed in Table 1.

[0085] Consequently, Twist1 mRNA expression in Huh7 cells treated with NSC-38270 decreased in a dose-dependent manner, indicating that NSC-38270 inhibits the transcription of Twist1 (Fig. 7).

[0086] In addition, mRNA expression of CDH1 in Huh7 cells treated with NSC-38270 was induced in a dose-dependent manner (Fig. 7).

[0087] In summary, this suggests that NSC-38270 inhibits migration and invasion by regulating Twist1 protein levels through the inhibition of Twist1 gene expression in Huh7 cells, thereby inducing CDH1 expression.

[0088]

[0090] Example 5: Analysis of cell growth in Huh7 cells treated with NSC-38270

[0091] To analyze the effects of NSC-38270 on cell division and growth of Huh7 cells, a cell growth analysis was performed on Huh7 cells treated with NSC-38270.

[0092] Cells were seeded into 24-well plates at a density of 15%, cultured overnight, and then treated with various concentrations of NSC-38270 (5, 10, 20 nM). Cell viability was measured every 24 hours after treatment with NSC-38270 using the EZ-Cytox reagent. The baseline viability before NSC-38270 treatment (hour 0) was set to 1, and subsequent viability rates at different time points were compared and presented in a graph. The experiment was repeated three times.

[0093] As a result, cell growth gradually stopped as the concentration of NSC-38270 increased. After 48 hours, cell growth in the group treated with 20 nM NSC-38270 decreased 3.5-fold compared to the group not treated with NSC-38270 (Fig. 8).

[0095] Example 6: Analysis of morphological changes in Huh7 cells treated with NSC-38270

[0096] To analyze morphological changes in Huh7 cells treated with NSC-38270, cells were seeded at low density in 12-well plates. Cells were treated with NSC-38270, and random images were captured every 6 hours using a JuLI Stage real-time cell history recorder (NanoEnTek, Inc., Korea).

[0097] As a result, when treated with a low concentration (20 nM) of NSC-38270, cell growth was inhibited over time, and each cell tended to maintain a round shape. Conversely, when treated with a relatively high concentration (50 nM) of NSC-38270, the cells developed into a long, pointed shape within 6 hours. After 18 hours, the cells showed signs of gradual shrinkage, and after 24 hours, the cells appeared to clump together (Fig. 9).

[0098] The analysis results according to the present embodiment suggest that NSC-38270 induces apoptosis in HCC cell lines.

[0100] Example 7: Analysis of apoptosis in Huh7 cells treated with NSC-38270

[0101] 7-1. Analysis of Apoptosis Using Annexin V Staining

[0102] To determine whether NSC-38270 induces apoptosis, apoptotic cells were analyzed in Huh7 cells treated with various concentrations of NSC-38270 (20, 50 nM) for 24 hours.

[0103] Cells were seeded into 6-well plates and cultured overnight. Cells were treated with NSC-38270 for 24 hours and then washed twice with PBS. For the apoptosis assay, cells were mixed with Muse® Annexin V & Dead Cell Assay Reagent (Millipore, Burlington, MA, USA). The mixture was incubated at room temperature in the dark for 20 minutes and then evaluated using a Muse Cell Analyser (Millipore).

[0104] As a result, an analysis of apoptosis using Annexin V staining showed that NSC-38270 increased early and late apoptotic / dead cells in a dose-dependent manner, and the total number of cells that underwent apoptosis also increased significantly (Fig. 10).

[0106] 7-2. Analysis of Histone H2A.X Activation

[0107] We evaluated the activation of histone H2A.X by analyzing histone H2A.X phosphorylation, another apoptosis marker, to determine whether it induces apoptosis in Huh7 cells treated with NSC-38270.

[0108] Histone H2A.X phosphorylation assays were performed using the Guava® DNA Damage Histone H2A.X Dual Detector Kit (Millipore; FCCS025153). Cells were treated with NSC-38270 for 24 hours and then harvested using PBS. Subsequently, cells were fixed in fixation buffer on ice for 5 minutes and then washed with PBS. Permeation buffer was added to the fixed cells and incubated on ice for 5 minutes. After further washing with PBS, cells were incubated with anti-phospho-histone H2A.X (Ser139)-Alexa Fluor 555 and anti-histone H2A.X-PECy5-conjugated antibodies. The reaction was carried out in the dark at room temperature for 30 minutes. Finally, cells were resuspended in 1X assay buffer and analyzed using the Muse Cell Analyser (Millipore) according to the manufacturer's protocol.

[0109] As a result, when cells were treated with 10 nM NSC-38270, activated histone H2A.X increased by more than three times compared to the untreated condition. In addition, when treated with 50 nM NSC-38270, the proportion of cells with activated histone H2A.X was higher than the proportion of cells with inactivated histone H2A.X (Fig. 11).

[0111] 7-3. Confirmation of protein and mRNA expression levels of apoptosis-related genes

[0112] Western blot and real-time PCR were performed to determine the expression levels of apoptosis-related genes altered by NSC-38270-induced apoptosis in Huh7 cells.

[0113] To evaluate the protein levels of cleaved caspase-3 and cleaved PARP, which are apoptosis-related genes, a Western blot experiment identical to that in Example 4-1 was performed. As a result, the protein levels of cleaved caspase-3 and cleaved PARP increased in a dose-dependent manner upon treatment with NSC-38270 (Fig. 12).

[0114] In addition, to evaluate the mRNA levels of the apoptosis-related genes Bax and Bcl2, real-time PCR identical to that in Example 4-2 was performed. As a result, when cells were treated with NSC-38270 for 24 hours, the mRNA expression level of Bax increased in a dose-dependent manner, while the expression level of Bcl2 decreased (Fig. 13).

[0115] The analysis results according to the present embodiment suggest that NSC-38270 induces apoptosis in Huh7 cells.

[0117] Example 8: Effects of NSC-38270 on normal cells

[0118] 8-1. Analysis of Cell Growth in Normal Cells Treated with NSC-38270

[0119] One of the important evaluation factors in the development of anticancer drugs is that compounds such as NSC-38270 induce apoptosis in cancer cells without affecting normal cells. Accordingly, to evaluate the effect of NSC-38270 on the cell growth of normal cells such as THLE-2 and Chang hepatocytes, a cell growth analysis was performed in the same manner as in Example 5.

[0120] As a result, it was confirmed that both THLE-2 and Chang liver cells proliferated well when treated with NSC-38270 (Fig. 14).

[0122] 8-2. Analysis of Morphological Changes in Normal Cells Treated with NSC-38270

[0123] To analyze the morphological changes in THLE-2 and Chang liver cells treated with NSC-38270, experiments were performed in the same manner as in Example 6.

[0124] As a result, no morphological changes were detected in THLE-2 and Chang liver cells after treatment with NSC-38270 for 24 hours (Fig. 15). This is in contrast to the morphological changes found in Huh7 cells after treatment with NSC-38270 for 24 hours.

[0126] 8-3. Analysis of Apoptosis in Normal Cells Treated with NSC-38270 Using Annexin V Staining

[0127] To determine whether NSC-38270 induces apoptosis in normal cells, an apoptosis analysis was performed in the same manner as in Example 7-1.

[0128] Even when treated with 50 nM NSC-38270, which induces strong apoptosis in Huh7 cells, THLE-2 and Chang liver cells did not undergo any apoptosis (Fig. 16).

[0130] 8-4. Analysis of Histone H2A.X Activation in Normal Cells Treated with NSC-38270

[0131] To confirm DNA damage in normal cells treated with NSC-38270, histone H2A.X phosphorylation analysis was performed in the same manner as in Example 7-2.

[0132] The activity of histone H2A.X in THLE-2 and Chang liver cells was not altered upon treatment with NSC-38270 (Fig. 17).

[0133] According to the analysis results of this experimental example, it suggests that NSC-38270 can induce clear apoptosis in the liver cancer cell line Huh7, but not in normal liver cell lines.

Claims

Claim 1 A pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis comprising, as an active ingredient, a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof. [Chemical Formula 1] Claim 2 A pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis, characterized in that, in addition to the active ingredient in claim 1, it further comprises a pharmaceutically acceptable carrier, excipient, or diluent. Claim 3 A pharmaceutical composition for preventing and inhibiting metastasis of liver cancer cells, wherein, in claim 1, the active ingredient inhibits the migration, invasion, and both of the liver cancer cells. Claim 4 A pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis, characterized in that, in claim 1, the active ingredient inhibits epithelial-mesenchymal transition. Claim 5 A pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis, wherein, in claim 1, the active ingredient inhibits the expression of the Twist1 gene, which is an EMT-TF gene. Claim 6 A pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis, wherein, in claim 1, the active ingredient induces the expression of the E-cadherin1 (CDH1) gene. Claim 7 A pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis, characterized in that, in claim 1, the active ingredient does not affect the expression of the EMT-TF genes, namely Snail1 or Zeb1. Claim 8 A pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis, characterized in that, in claim 7, the EMT-TF gene is a Snail1 or Zeb1 gene. Claim 9 A pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis, characterized in that, in claim 1, the pharmaceutical composition further comprises an anticancer agent. Claim 10 A pharmaceutical composition for preventing and inhibiting liver cancer cell metastasis according to claim 9, characterized in that the anticancer agent is selected from the group consisting of oxaliplatin, pemetrexed, cisplatin, irinotecan, gemcitabine, carboplatin, bortezomib, fluorouracil (5-FU), cyclophosphamide, paclitaxel, vincristine, etoposide, and doxorubicin. Claim 11 A health food composition for preventing and inhibiting liver cancer cell metastasis comprising a compound represented by the following chemical formula 1 as an active ingredient. [Chemical Formula 1] Claim 12 1) A step of treating a liver cancer cell line with a compound represented by Chemical Formula 1 as a positive control and measuring the Twist1 mRNA or protein level; [Chemical Formula 1] 2) a step of treating a liver cancer cell line with a candidate substance and measuring the level of Twist1 mRNA or protein; 3) a step of determining that the candidate substance is a substance having liver cancer cell metastasis inhibitory activity if the measurement result of step 2) is lower than the measurement result of step 1); comprising a screening method for a substance having liver cancer cell metastasis inhibitory activity. Claim 13 A screening method for a substance having liver cancer cell metastasis inhibitory activity, wherein, in claim 12, after step 3), the step of determining whether the candidate substance increases the expression of the E-cadherin1 (CDH1) gene.

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