Pharmaceutical composition for preventing or treating ovarian cancer comprising gentian violet

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of ovarian cancer using gentian violet as an active ingredient. More specifically, it is confirmed that gentian violet can be utilized as an ovarian cancer treatment by inducing the cleavage of PARP or Caspase-3 in ovarian cancer cells and increasing apoptosis and ROS as it increases the expression of p53, PUMA (p53-upregulated modulator of apoptosis), BAX (BCL2 associated X), or p21.

Description

Technology Field

[0001] The present invention provides a pharmaceutical composition for the prevention or treatment of ovarian cancer, comprising gentian violet as an active ingredient. Background Technology

[0002] Gentian violet, also known as crystal violet, was used as an antibacterial and antifungal agent until the discovery of penicillin. Currently, gentian violet is used to stain bacterial cells to distinguish between Gram-positive and Gram-negative bacteria, and recently, it is being studied as a treatment for skin diseases, anti-angiogenesis, and anti-tumor.

[0003] Ovarian cancer accounts for 3.4% of cancers in women and ranks eighth in incidence worldwide. It is the fifth leading cause of cancer-related deaths in women, following lung, breast, colon, and pancreatic cancers. In the United States, it is estimated that 19,880 new cases of ovarian cancer were diagnosed in 2022, with an estimated 12,810 deaths. In particular, because there are no specific symptoms in the early stages of ovarian cancer and there are no effective diagnostic methods or specific markers, most patients are often discovered at stage 3 or 4, when the cancer has already progressed.

[0004] Therefore, research on therapeutic agents is necessary for the effective treatment of ovarian cancer. Prior art literature

[0005] 1. U.S. Patent Publication No. 2022-0072089 (Published March 10, 2022) The problem to be solved

[0006] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of ovarian cancer comprising gentian violet, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0007] Another objective of the present invention is to provide a composition for increasing p53, PUMA, BAX, or p21 expression, comprising gentian violet, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. means of solving the problem

[0008] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of ovarian cancer comprising gentian violet, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] In addition, the present invention provides a composition for increasing p53, PUMA, BAX, or p21 expression, comprising gentian violet, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. Effects of the invention

[0010] The present invention relates to a pharmaceutical composition for the prevention or treatment of ovarian cancer using gentian violet as an active ingredient. More specifically, it is confirmed that gentian violet can be utilized as an ovarian cancer treatment by inducing the cleavage of PARP or Caspase-3 in ovarian cancer cells and increasing apoptosis and ROS as it increases the expression of p53, PUMA (p53-upregulated modulator of apoptosis), BAX (BCL2 associated X), or p21. Brief explanation of the drawing

[0011] Figure 1 is the chemical formula structure of gentian violet. Figure 2 shows the results confirming the inhibitory effect on ovarian cancer cell proliferation by gentian violet treatment. Figure 3 shows the results of confirming the cell death induction effect in ovarian cancer cells by gentian violet treatment. Figure 4 shows the results of confirming the effect of gentian violet treatment on inducing the cleavage of PARP and Caspase-3 in ovarian cancer cells. Figure 5 shows the results of confirming the effect of increasing ROS in ovarian cancer cells by gentian violet treatment. Figure 6 shows the results of confirming the effect of increasing the expression of p53, PUMA, BAX, and p21 in ovarian cancer cells by gentian violet treatment. Figure 7 shows the results of confirming the inhibitory effect on cell proliferation in human venous endothelial cells and ovarian surface epithelial cells by gentian violet treatment. Figure 8 shows the results of comparing the inhibitory effect on proliferation of breast, colorectal, and lung cancer cells by gentian violet treatment. Specific details for implementing the invention

[0012] The present invention will be described in more detail below.

[0014] The present invention provides a pharmaceutical composition for the prevention or treatment of ovarian cancer comprising gentian violet, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0015] The above pharmaceutical composition can increase apoptosis and ROS in ovarian cancer cells.

[0016] The above pharmaceutical composition can induce cleavage of PARP or Caspase-3.

[0017] The above pharmaceutical composition can increase the expression of p53, PUMA (p53-upregulated modulator of apoptosis), BAX (BCL2 associated X), or p21.

[0018] The above pharmaceutical composition may contain 0.01 to 50 parts by weight of gentian violet per 100 parts by weight of the total pharmaceutical composition.

[0020] The gentian violet of the present invention is represented by Chemical Formula 1, and its chemical structural formula is C 25 H 30 It is ClN3 .

[0021] [Chemical Formula 1]

[0022]

[0023] The above pharmaceutical composition may be any one dosage form selected from the group consisting of tablets, capsules, injections, troches, powders, granules, liquids, suspensions, oral liquids, emulsions, syrups, suppositories, vaginal tablets, and pills, but is not limited thereto.

[0024] In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, leavening agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0025] Specifically, the carrier, excipient, and diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups, and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as the base for suppositories.

[0026] According to one embodiment of the present invention, the pharmaceutical composition may be administered to a subject in a conventional manner through an intravenous, intra-arterial, intra-abdominal, intramuscular, intra-arterial, intra-abdominal, intrasternal, transdermal, nasal, inhalation, local, rectal, oral, ocular, or intradermal route.

[0027] The dosage of the active ingredient according to the present invention may vary depending on the subject's condition and body weight, the type and severity of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by a person skilled in the art. The daily dosage may be 0.01 mg / kg to 200 mg / kg, preferably 0.1 mg / kg to 200 mg / kg, and more preferably 0.1 mg / kg to 100 mg / kg. The administration may be performed once a day or divided into several doses, and the scope of the present invention is not limited by this.

[0029] In addition, the present invention provides a composition for increasing p53, PUMA, BAX, or p21 expression, comprising gentian violet, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0031] Hereinafter, to aid in understanding the present invention, examples and the like will be described in detail. However, the following examples and the like are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples and the like. The examples and the like of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0033] <Experimental Example 1> Cell Culture

[0034] Human epithelial ovarian adenocarcinoma cell lines SKOV3 and A2780 were purchased from KBCC (Korea Biotechnology Commercialization Center, Incheon, Korea), and OVCAR8, MDA-MB-231, HCT116, A549, HUVEC, and HIO-80 were purchased from the American Type Culture Collection (Rockville, MD, USA). OVCAR8, SKOV3, A2780, HCT116, and A549 were cultured in Roswell Park Memorial Institute medium (Thermo Fisher Scientific; Waltham, MA, USA) containing 10% heat-inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin. MDA-MB-231 was cultured in Dulbecco's Modified Eagle's Medium (Gibco, Carlsbad, CA, USA) containing 10% heat-inactivated FBS (Gibco, Carlsbad, CA, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin. HUVEC was cultured in Endothelial cell growth Basal Medium-2 containing 10% heat-inactivated FBS, hydrocortisone, hFGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF, GA-1000, and HEPARIN. HIO-80 was cultured in M199 and MDCB 105 (1:1) (Thermo Fisher Scientific; Waltham, MA, USA) media containing 4% heat-inactivated FBS (Gibco, Carlsbad, CA, USA) and 7.5 μg / mL insulin. All cells were cultured in an incubator at 37°C, 5% CO2, and in a humid environment.

[0036] <Experimental Example 2> Cell Proliferation Analysis

[0037] Gentian violet (hereinafter referred to as GV) was purchased from TargetMol (Boston, MA, USA). Cell proliferation was measured according to the analysis method of the WST-1 Cell Proliferation Assay Kit (Takara, Kyoto, Japan). 5 × 10⁶ in 200 μl of culture medium. 3 A number of OVCAR8 and A2780 cells or 2 × 10⁶ 3 A specified number of SKOV3 cells were seeded into 96-well tissue culture plates, and after 24 hours, the cells were treated with GVs of 0, 0.3, 1, or 3 μM. After incubating for 24, 48, and 72 hours, 20 μl of WST-1 reagent was added to each well, and the cells were incubated for an additional hour at 37°C. The percentage of viable cells was determined by measuring the absorbance of the formazan product at 450 nm using a microplate reader (Synergy H1, BioTek, Winooski, VT, USA). IC 50 The values ​​were calculated by fitting the dose-response curve to a 4-parameter variable slope S-shaped dose-response model in GraphPad Prism 5.0 software (GraphPad software Inc., San Diego, CA, USA).

[0039] <Experimental Example 3> TUNEL Analysis

[0040] TUNEL analysis was performed by slightly modifying the analysis method of the in situ cell death detection kit, fluorescein (Roche, Meylan, France). 4 × 10 4A number of OVCAR8 and SKOV3 cells were seeded onto 8-well chamber slides (Thermo Fisher Scientific) coated with 1 μg / ml poly-L-lysine (Sig-ma-Aldrich, St. Louis, MO, USA), and after 24 hours, the cells were treated with 0, 0.3, 1, or 3 μM GV. After incubating for 24 hours, the cells were fixed by treating them with 4% paraformaldehyde for 10 minutes, and cell permeability was increased with 0.1% Triton X-100 for 3 minutes at room temperature. Subsequently, the cells were stained with 100 μl of TUNEL-reaction mixture at 37°C for 1 hour, followed by staining with Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) for 3 minutes. Slides were washed twice with PBS, treated with mounting medium (Vector Laboratories, Burlingame, CA, USA), and observed using a confocal laser scanning microscope (Leica, Wetzlar, Germany). Cell TUNEL fluorescence was quantified using Image J software (NIH, Bethesda, MD, USA), and TUNEL fluorescence intensity was calculated by dividing the cell death fluorescence intensity by the control group.

[0042] <Experimental Example 4> Western Blot Analysis

[0043] 2 x 10 in a 6-well plate 5A number of OVCAR8 and SKOV3 cells were lysed in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Thermo Fisher Scientific), and the total protein amount was calculated using the BCA Protein Assay Kit (Thermo Fisher Scientific). 10 μg of protein samples from the cell lysates of OVCAR8 and SKOV3 were separated by 12% SDS-PAGE and transferred to nitro-cellulose membranes (Amersham, Little Chalfont, UK). Block the membrane with 5% bovine serum albumin in Tris-buffered saline plus 0.1% (v / v) Tween 20 (TBST) at room temperature for 1 hour, and PARP (1:1000; #9542), cleaved PARP (1:1000; #5625), caspase-3 (1:1000; #14220), cleaved caspase-3 (1:200; #9664), p53 (1:1000; #2527), PUMA (1:1000); Primary antibodies #12450), BAX(1:1000; #5023), p21(1:1000, #2947)(Cell Signaling Technology, Danvers, MA, USA) and β-actin(1:1000, #sc-47778)(Santa Cruz, Heidelberg, Germany) were attached overnight at 4°C. After washing 6 times with TBST, the membrane was attached with HPB (horseradish peroxidase-conjugate) secondary antibodies for one hour at room temperature.Afterward, the membrane was washed 6 times with TBST, chemiluminescence was induced using ECL (enhanced chemiluminescence; Thermo Fisher Scientific), and images were captured using an Amersham ImageQuant 800 (Cytiva, Logan, UT, USA). Protein expression intensity was analyzed using ImageJ (National Institutes of Health; https: / / imagej.nih.gov / ij / ).

[0045] <Experimental Example 5> Measurement and Analysis of Reactive Oxygen Species (ROS)

[0046] 2 × 10 5 OVCAR8 and SKOV3 cells were seeded into a 6-well plate, and after 24 hours, the cells were treated with GVs of 0, 0.18, 0.37, 0.75, 1.5, or 3 μM. After 24 hours of incubation, the cells were washed with PBS, and 10 μM H2DCFDA (Invitrogen) was added and incubated at 37°C for 30 minutes. Subsequently, the cells were washed three times with PBS, lysed with RIPA buffer, and ROS generation was measured at 485 / 530 nm using a microplate reader.

[0048] <Example 1> Chemical structure of gentian violet

[0049] The compound used in the present invention is gentian violet. The chemical structural formula of gentian violet is C 25 H 30 It is ClN3, a triarylmethane dye used for histological staining and Gram staining for bacterial classification.

[0051] <Example 2> Confirmation of the inhibitory effect of gentian violet on ovarian cancer cell proliferation

[0052] To determine whether GV inhibits the proliferation of ovarian cancer cells, OVCAR8, SKOV3, or A2780 cells were seeded into 96-well plates, treated with indicated concentrations of GV for 72 hours, and cell growth was measured using the WST-1 cell proliferation assay. Additionally, OVCAR8, SKOV3, or A2780 cells were treated with 0, 0.33, 1, or 3 μM of GV for 0, 24, 48, or 72 hours. Relative cell proliferation rates were calculated by measuring absorbance at 450 nm using the WST-1 assay to determine mitochondrial succinate reductase activity. Data were expressed as the mean ± SEM of the overlaps and presented from one of two independent experiments.

[0053] As a result, according to Fig. 2, IC 50 By confirming that the values ​​were 0.665, 0.5867, and 1.0367 μM, respectively, it was confirmed that GV inhibits proliferation in all three cell lines as the concentration increases. In particular, it was found that growth was completely inhibited in all three ovarian cancer cell lines when treated with 3 μM GV.

[0054] Through this, GV was able to demonstrate its potential as an ovarian cancer treatment by inhibiting the proliferation of ovarian cancer cells in a dose- and time-dependent manner.

[0056] <Example 3> Confirmation of whether gentian violet induces apoptosis in ovarian cancer cells

[0057] To determine whether the cell growth inhibitory effect of GV in ovarian cancer cells is due to apoptosis, we first conducted a TUNEL analysis to check for DNA fragmentation.

[0058] OVCAR8 and SKOV3 were treated with 0, 1, or 3 μM GV for 48 hours, and images were acquired using a confocal microscope. Nuclei of TUNEL-positive cells were shown in green, and nuclei of total cells stained with Hoechst 33342 were shown in blue; relative TUNEL fluorescence intensity was quantified across three randomly selected microscope fields from the negative controls. Data are expressed as mean ± SEM and represent one of two independent experiments (bar = 100 μm).

[0059] As a result, according to Figure 3, it was confirmed that the TUNEL positive distribution in both OVCAR8 and SKOV3 cells increased when GV was treated, unlike the negative control.

[0060] In addition, OVCAR8 and SKOV3 were treated with 0, 1, or 3 μM GV for 24 hours, and whole cell lysates were subjected to Western blot with Caspase-3, cleaved-Caspase-3, PARP, cleaved-PARP, or β-actin antibodies.

[0061] As a result, according to Figure 4, Western blot analysis revealed that GV induces the cleavage of Caspase-3 and PARP.

[0062] Through this, it was found that GV induces apoptosis in ovarian cancer cells by inducing the cleavage of Caspase-3 and PARP.

[0064] <Example 4> Confirmation of whether gentian violet increases ROS in ovarian cancer cells

[0065] To investigate the effect of GV on ROS changes in ovarian cancer cells, OVCAR8 and SKOV3 cells were treated with the indicated concentrations of GV for 24 hours and stained with 10 μM H2DCFDA. ROS was measured at 485 / 530 nm using a microplate reader. Data were presented as the mean ± SEM of overlaps and one of two independent experiments was selected.

[0066] As a result, according to Figure 5, it was confirmed that GV significantly increased ROS in both OVCAR8 and SKOV3 cells as the dose increased.

[0067] Additionally, to investigate molecules of the ROS downstream, OVCAR8 and SKOV3 were treated with 0, 1, or 3 μM GV for 24 hours, and whole cell lysates were subjected to Western blot with antibodies against p53, PUMA, BAX, p21, or β-actin. The results were presented from a selection of at least three independent experiments.

[0068] As a result, according to Figure 6, it was found that the amounts of p53, PUMA, BAX, and p21 proteins were significantly increased when treated with GV.

[0069] Through this, it was confirmed that GV induces apoptosis in ovarian cancer cells by increasing cellular ROS and inducing an increase in the amount of downstream proteins.

[0071] <Example 5> Confirmation of the cell proliferation inhibitory effect of gentian violet on non-tumor cells and other cell lines

[0072] 0.5x10 4 A number of HUVEC and HIO-80 cells were seeded into a 96-well plate, and after 24 hours, GV was treated at the concentrations shown in Fig. 7. After culturing the cells for 72 hours, 20 μl of WST-1 was added to each well and incubated at 37°C for one hour. Subsequently, the absorbance was measured at 450 nm using a microplate reader.

[0073] As a result, according to Fig. 7, GV showed cell proliferation inhibitory efficacy IC in non-tumor cells HUVEC and HIO-80. 50 With values ​​of 4.62 μM and 2.37 μM, respectively, the IC50 values ​​in ovarian cancer cell lines were 50 It was confirmed that GV has a specific anticancer effect on ovarian cancer cells by confirming that it is about 4-7 times higher than the value of 0.6 μM.

[0074] In addition, to determine whether GV has an inhibitory effect on cell proliferation in breast, colorectal, and lung cancers, excluding ovarian cancer, 0.5 x 10 4 A number of MDA-MB-231, HCT116, and A549 cells were seeded into a 96-well plate, and after 24 hours, GV was treated at the concentrations shown in Fig. 8. After culturing the cells for 72 hours, 20 μl of WST-1 was added to each well and incubated for one hour. Subsequently, the absorbance was measured at 450 nm using a microplate reader.

[0075] As a result, according to Figure 8, it was confirmed that GV did not have a distinct inhibitory effect on cell proliferation in MDA-MB-231. Cell proliferation inhibitory efficacy IC of HCT116 and A549 50 The values ​​were 2.09 μM and 1.76 μM, respectively, which were about 2-3 times higher than the value of 0.6 μM in ovarian cancer cell lines, indicating that GV has a greater anticancer effect on ovarian cancer cell lines compared to breast, colorectal, and lung cancer cell lines.

[0077] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0078] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of ovarian cancer, wherein the pharmaceutical composition comprises gentian violet or a pharmaceutically acceptable salt thereof as an active ingredient, and the pharmaceutical composition is characterized by increasing the expression of p53, PUMA (p53-upregulated modulator of apoptosis), BAX (BCL2 associated X), or p21. Claim 2 A pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition increases apoptosis and ROS in ovarian cancer cells. Claim 3 A pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition induces the cleavage of PARP or Caspase-3. Claim 4 delete Claim 5 A pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition comprises 0.01 to 50 parts by weight of gentian violet per 100 parts by weight of the total pharmaceutical composition. Claim 6 A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is any one formulation selected from the group consisting of tablets, capsules, injections, troches, powders, granules, liquids, suspensions, oral liquids, emulsions, syrups, suppositories, vaginal tablets, and pills. Claim 7 A reagent composition for increasing p53, PUMA, BAX, or p21 expression, comprising gentian violet or a pharmaceutically acceptable salt thereof as an active ingredient.

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