KR20230000108A - Pharmaceutical composition for preventing or treating lung cancer comprising diethylstilbestrol - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating lung cancer containing diethylstilbestrol as an active ingredient, and the pharmaceutical composition containing diethylstilbestrol suppresses the expression of ANO1 to have a useful effect as the pharmaceutical composition for preventing or treating lung cancer. In addition, the pharmaceutical composition containing diethylstilbestrol of the present invention inhibits the survival and migration of PC9 cells and induces apoptosis, while the composition does not affect the survival of hepatocytes, thereby being useful as the pharmaceutical composition for inhibiting lung cancer development and metastasis.

Description

Pharmaceutical composition for preventing or treating lung cancer comprising diethylstilbestrol

The present invention relates to a pharmaceutical composition for preventing or treating lung cancer containing diethylstilbestrol (DES) as an active ingredient.

Lung cancer is classified into two types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Among them, NSCLC is the most prominent cause of cancer death, accounting for more than 80% of lung cancer cases. Therefore, several attempts have been made to increase treatment efficiency by using molecular or immunological methods to treat lung cancer through targeted therapy or immunotherapy. Some therapies are targeting EGFR-TKIs and PD-1 with various therapeutic targets, while others inhibit abnormal signaling, including the PI3K/AKT/mTOR pathway, the RAS/BRAF/MAPK pathway, and the JAK/STAT pathway.

However, the pathological mechanism of non-small cell lung cancer (NSCLC) remains unclear, and limitations to non-small cell lung cancer treatment are becoming prominent due to mutations in treatment targets and resistance to therapeutic drugs. As a result, efforts to develop new therapeutic agents and find new therapeutic targets for the treatment of non-small cell lung cancer are required.

Chlorine ion channel (CaCC), one of the anion channels considered as therapeutic targets for various carcinomas, is activated by calcium and is known to regulate epithelial cell secretion and cell volume. Recently, it has been known that ANO1 (Anoctamin1), a major CaCC, is overexpressed in cancer cells of the stomach, prostate, head and neck, breast and colon (non-patent literature, Britschgi A, et al., Proc. Natl Acad. Sci. USA 2013, 110 , E1026-E1034). ANO1 is involved in cancer development by promoting tumor-inducing signals such as the EGFR and CAMK pathways, which are major targets for lung cancer treatment. In particular, ANO1 and EGFR are involved in cancer development by forming a functional complex that promotes cancer development of head and neck cancer.

ANO1 can be an important target for the treatment of various cancers, but the pathological mechanisms of ANO1 in non-small cell lung cancer have not been precisely identified. The development of various treatments for non-small cell lung cancer continues, and anticancer drug candidates with mechanisms that inhibit ANO1 have not been developed for the treatment of non-small cell lung cancer.

The present inventors found that diethylstilbestrol is an ANO1 specific inhibitor, and completed the invention through further research.

An object of the present invention is to provide a preventive or therapeutic agent for lung cancer, particularly non-small cell lung cancer (NSCLC).

Another object of the present invention is to provide an inhibitor of metastasis of lung cancer, particularly non-small cell lung cancer (NSCLC).

In order to achieve the above purpose,

The present invention provides a pharmaceutical composition for preventing or treating lung cancer containing diethylstilbestrol or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for inhibiting metastasis of lung cancer containing diethylstilbestrol or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for inhibiting ANO1 expression containing diethylstilbestrol or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition containing diethylstilbestrol of the present invention inhibits the proliferation of lung cancer cells while inhibiting ANO1, has excellent anticancer activity against lung cancer, and has an effect of inhibiting metastasis of lung cancer.

1A is a diagram showing the measurement of ANO1-induced current in HEK293T cells expressing ANO1.
Figure 1b is a diagram showing the density of ANO1-induced current in HEK293T cells expressing ANO1.
Figure 2a is a diagram showing the expression level of ANO1 in rat thyroid cells FRT and FRT-hANO1 (cells artificially overexpressing ANO1) and lung cancer cell lines A549, H1993, HCC827, H1975, and PC9 cells.
2B is a diagram showing relative ANO1 protein levels in FRT, FRT-hANO1, and lung cancer cell lines A549, H1993, HCC827, H1975, and PC9 cells.
Figure 2c is a diagram showing the expression levels of ANO1 by Eact, Ani9, and diethylstilbestrol in lung cancer cell line PC9 cells.
Figure 2d is a diagram showing the expression levels of ANO1 by Eact, Ani9, and diethylstilbestrol in PC9 cells, a lung cancer cell line, normalized to beta-actin.
Figure 3a is a diagram showing cell viability by Eact, Ani9, and diethylstilbestrol in lung cancer cell line H1975 cells.
Figure 3b is a diagram showing cell viability by Eact, Ani9, and diethylstilbestrol in lung cancer cell line PC9 cells.
Figure 3c is a view showing the results of performing a cell migration assay (migration assay) by treating lung cancer cell line PC9 cells with diethylstilbestrol.
Figure 3d is a view showing the degree of cell migration inhibition effect by treating the lung cancer cell line PC9 cells with diethylstilbestrol and Ani9.
Figure 4a is a diagram showing the activity of Caspase3 in PC9 cells, a lung cancer cell line treated with Eact, Ani9, and diethylstilbestrol.
Figure 4b is a diagram showing the activity of Cleaved PARP-1 in PC9 cells, a lung cancer cell line treated with Eact, Ani9, and diethylstilbestrol.
Figure 4c is a diagram showing cell viability by Eact, Ani9, and diethylstilbestrol in HepG2 (hepatocyte) hepatocytes.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention provides a pharmaceutical composition for preventing or treating lung cancer containing diethylstilbestrol or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for inhibiting metastasis of lung cancer containing diethylstilbestrol or a pharmaceutically acceptable salt thereof as an active ingredient.

The diethylstilbestrol may be represented by Formula 1 below.

[Formula 1]

The IUPAC name of diethylstilbestrol is 4,4`-(3E)-hex-3-ene-3,4-diyldiphenol, (E)-3,4-bis(4-hydroxyphenyl)- Also called 3-hexene, abbreviated DES.

In another aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of ANO1 or the activity of ANO1 containing diethylstilbestrol or a pharmaceutically acceptable salt thereof as an active ingredient.

Diethylstilbestrol or a pharmaceutically acceptable salt thereof may induce apoptosis of lung cancer cells or inhibit migration of lung cancer cells. As an example, the lung cancer cells may be lung cancer cells generated due to overexpression of ANO1.

In the present invention, the lung cancer may be non-small cell lung cancer or lung cancer caused by overexpression of ANO1.

Diethylstilbestrol inhibits Ca ^{2+ -activated} Cl -channel ⁽ CaCC; Ca ²⁺ -activated Cl -channel) and induces apoptosis. Specifically, diethylstilbestrol inhibits ANO1, inhibits the growth and migration of cancer cells, and induces apoptosis.

In one embodiment of the present invention, it was confirmed that diethylstilbestrol and Ani9, known as an inhibitor of ANO1, suppress ANO1-induced current in HEK293T cells expressing ANO1 (see FIGS. 1a and 1b), and PC9 among lung cancer cell lines. ANO1 is overexpressed in cells (see Figs. 2a and 2b), and compared to Eat, known as an activator of ANO1, and Ani9, known as an inhibitor of ANO1, it is confirmed that diethylstilbestrol reduces ANO1 protein expression ( see Figures 2c and 2d).

In another embodiment of the present invention, when Eact, Ani9, and diethylstilbestrol were treated with H1975 cells, in which ANO1 is less expressed among lung cancer cell lines, it was confirmed that there was almost no change in cell viability (see FIG. 3a), lung cancer Unlike Eact or Ani9 in PC9 cells, which overexpress ANO1 among cell lines, it was confirmed that the cell viability decreased only when diethylstilbestrol was treated (see FIG. 3b). In addition, through cell migration analysis, it was confirmed that the migration of PC9 cells was reduced by diethylstilbestrol (see Figs. 3c and 3d).

In another embodiment of the present invention, unlike Eact or Ani9 in PC9 cells, when cells are treated with diethylstilbestrol, Capase3 and Cleaved PARP-1 activities are significantly increased, confirming that apoptosis is induced. (See Figures 4a and 4b). In addition, it was confirmed that diethylstilbestrol had no effect on the cell viability of HepG2 hepatocytes (see FIG. 4c).

Therefore, the pharmaceutical composition containing diethylstilbestrol of the present invention induces apoptosis of cancer cells, does not weaken the viability of hepatocytes, and reduces the growth and migration of cancer cells, thereby preventing or treating cancer. It is useful as a pharmaceutical composition for

In the present invention, "treatment" refers to all activities in which symptoms are improved or beneficially changed by administration of the composition according to the present invention.

In the present invention, "prevention" refers to any action that suppresses or delays the onset of disease by administering the composition according to the present invention.

In the present invention, "metastasis" refers to a phenomenon in which tumor cells move to a place separated by a distance from one organ or part, and "metastasis inhibition" fundamentally blocks the occurrence of metastasis by administration of the composition according to the present invention. or any action that inhibits or delays the transition phenomenon.

The diethylstilbestrol may be used in the form of a pharmaceutically acceptable salt, and a pharmaceutically acceptable metal salt may be prepared using a base. An alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. In addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).

The diethylstilbestrol includes solvates, stereoisomers, hydrates and the like that can be prepared therefrom.

The pharmaceutical composition of the present invention can be used as a single agent, and can be prepared and used as a combination preparation by further including an approved drug. It can be formulated into a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient, or diluent.

In the present invention, "pharmaceutically acceptable" means that it does not significantly stimulate living organisms and does not inhibit the biological activity and properties of the active substance administered.

In the present invention, the pharmaceutical composition comprising a pharmaceutically acceptable carrier is a tablet, pill, powder, granule, capsule, suspension, internal solution, emulsion, syrup, sterilized aqueous solution, non-aqueous solvent, suspension, emulsion, It may have any one formulation selected from the group consisting of lyophilized preparations and suppositories.

The pharmaceutical composition may be in various oral or parenteral formulations. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, 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 suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, etc. may be used, but is not limited thereto.

In the composition of the present invention, diethylstilbestrol may be included in a pharmaceutically effective amount. "Pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's health condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

In another aspect, the present invention provides a method for preventing or treating lung cancer by administering a pharmaceutically effective amount of diethylstilbestrol or a pharmaceutically acceptable salt thereof to a subject.

The present invention provides a method for inhibiting the growth and metastasis of lung cancer by administering a pharmaceutically effective amount of diethylstilbestrol or a pharmaceutically acceptable salt thereof to a subject.

The present invention provides a method of inhibiting the activity or expression of ANO1 by administering a pharmaceutically effective amount of diethylstilbestrol or a pharmaceutically acceptable salt thereof to a subject.

In another aspect, the present invention provides the use of diethylstilbestrol or a pharmaceutically acceptable salt thereof for the preparation of a drug for preventing or treating lung cancer.

The present invention provides the use of diethylstilbestrol or a pharmaceutically acceptable salt thereof for the preparation of a drug for inhibiting metastasis of lung cancer.

The present invention provides the use of diethylstilbestrol or a pharmaceutically acceptable salt thereof for the preparation of a drug for inhibiting the activity or expression of ANO1.

In the method or use, the detailed description of the pharmaceutical composition described above can be applied.

Hereinafter, the present invention will be described in detail by means of experimental examples.

However, the following experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following experimental examples.

<Compound Preparation>

For the experiment, diethylstilbestrol, a known one-activator of ANO1, and a known one-activator of ANO1 were prepared as shown in Table 1 below. In the description or drawings, a known activator of ANO1 is indicated by 'Eact', a known inhibitor of ANO1 by 'Ani9', and diethylstilbestrol is also indicated by DES.

division chemical structure where to buy DES

Sigma-Aldrich Eact

Tocris Bioscience Ani9

Tocris Bioscience

<Experiment protocol>

Details for carrying out Experimental Examples 1 to 7 of the present invention follow the protocols 1 to 9 below.

1. Cell culture

Fisher rat thyroid (FRT) cells stably expressing ANO1 and CFTR were provided by Alan Verkman (University of California, San Francisco, CA). FRT cells were cultured in Coon's modified F12 medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. HEK293T, A549, H1993, HCC827, H1975, and PC9 cells were provided by the Daegu-Gyeongbuk Medical Industry Promotion Foundation New Drug Development Center (DGMIF, Korea). HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), while the other cells were cultured in RPMI 1640 medium, supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin, respectively.

2. Whole-cell patch clamp experiments

Patch clamp recordings were performed on HEK-293 cells transiently expressing human ANO1. The pipette solution contains 150 mM N-methyl-D-glucamine-Cl (NMDG-Cl), 1 mM MgCl ₂ , 3 mM MgATP, 5 mM HEPES, 10 mM ethylene glycol tetraacetic acid (EGTA) and 6.6 mM CaCl ₂ (pH 7.2) (300 nM Ca ²⁺ ). The test tube solution consisted of 150 mM NMGD-Cl, 1 mM CaCl ₂ , 1 mM MgCl ₂ , 5 mM glucose and 10 mM HEPES (pH 7.4). Pipettes were prepared using borosilicate glass and had a resistance of 3-5 MΩ after fire polishing. The seal resistance was 3 to 10 GΩ. Pulse protocol: holding potential, 0 mV; ramp pulse, -100 to 100 mV; pulse duration, 1 s; and pulse interval, 20 sec. Recordings were performed at room temperature (RT) using an Axopatch 200 B (Axon Instruments, San Jose, CA, USA). Currents were digitized using a Digidata 1440A transducer (Axon Instruments), data filtered at 5 kHz and sampled at 1 kHz.

3. Western blot analysis

Protein samples were separated using 4%–20% Mini-PROTEIN TGXTM Precast Gels and transferred to nitrocellulose membranes (0.45 μm) using the Trans-Blot Turbo Blotting System (Bio-Rad). Blocking was performed using Everyblot blocking buffer (Bio-Rad). Subsequently, the membrane was incubated with recombinant Anti-TMEM16A antibody [SP31] (ab64085) (Abcam), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody #9101 (Cell signaling), p44/42 MAPK (Erk1/42 MAPK) 2) Antibody #9102 (Cell signaling), Phospho-EGF receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777 (Cell signaling), Anti-EGFR antibody (A-10) ｜SCBT - (Santa Cruz Biotechnology) and anti Incubated overnight with primary antibody containing -β-actin (Santa Cruz Biotechnology). Then, the membrane was incubated with HRP-conjugated anti-secondary IgG antibody (Enzo Life Science) for 1 hour at room temperature. Finally, visualization was performed on the ClarityTM Western ECL substrate using ChemiDoc (Bio-Rad).

4. Cell viability measurement

Cell viability assay was performed using Cell Counting Kit-8 (Dojindo, Rockville, MD, USA). PC9 and H1975 cells (2 x 10 ³ cells/well) were cultured in 96-well plates. Cell cultures were then re-incubated with medium (2.5% FBS) containing 7 concentrations of the compounds diluted ∼3-fold every 24 hours for 3 days. After 72 hours, CCK-8 solution was added and cells were further cultured for 2 hours. Absorbance was measured at 450 nm using a microplate reader (SynergyTM Neo, BioTek).

5. Cell migration assay

PC9 cells were cultured overnight at a density of 2×10 ⁴ cells/well to 90-100% confluence in 96-well ImageLock tissue culture plates (Essen BioScience). Monolayers were wounded according to the manufacturer's instructions. Cells were washed twice with PBS to remove detached cells, and then treated with the compound once every 24 hours with media containing the compound at the indicated concentrations (2.5% FBS) and cultured for 2 days. Images were then taken using a microscope. The data were analyzed by assessing the migration area of the cells, and the percent inhibition of cell migration was calculated using ImageJ software.

6. Caspase-3/CPP32 Colorimetric assay

Caspase-3 activity was measured according to the manufacturer's instructions (# K106, Biovison, Milpitas, CA, USA). PC9 cells were cultured in 6x-well plates until reaching 80% well area. Cells were treated with diethylstilbestrol, Eact and Ani9 for 24 hours, and 5x10 ⁶ cells were lysed using cell lysis buffer for 10 minutes on ice. After the cells were centrifuged at 4° C. for 5 minutes, the supernatant was separated. 100 μg protein/50 μL buffer was poured into each well with 2x reaction buffer containing 10 mM DTT. To measure Caspase3 activity, 5 μL of DEVD-pNA substrate was injected and incubated at 37°C for 1 hour. Optical density was measured at 400 nm using a microplate reader (SynergyTM Neo, BioTek).

7. Human Cleaved PARP-1 activity assay

Cleaved PARP-1 activity was measured according to the manufacturer's instructions (# ab174441, Abcam, UK). PC9 cells were cultured in 6x well plates until reaching 70% well area. Cells were treated with diethylstilbestrol, Eact and Ani9, and 24 hours later, 5x10 ⁷ cells were lysed using cell extraction buffer for 20 minutes on ice. After the cells were centrifuged at 4° C. for 20 minutes, the supernatant was separated. Then, 100 μg protein/50 μL buffer was poured into each well together with an antibody cocktail containing capture antibody and detection antibody and incubated for 1 hour. Wells were washed with 1x wash buffer and then TMB development solution was added for 10 minutes. Finally, stop solution was added and the optical density was measured at 450 nm using a microplate reader (SynergyTM Neo, BioTek).

8. Hepatocytotoxicity measurement

HepG2 cells were cultured in a black and clear bottom 96-well plate in medium (DMEM high glucose medium with 10% (v/v) FBS, 100 μg/mL penicillin and streptomycin) and the cells were approximately 70-80% were incubated at 37°C with 5% CO ₂ until reaching an area of . Test compounds were diluted using assay medium at concentrations of 0.01, 0.1, 1, 10 and 100 μM in 0.2% (v/v) DMSO solvent. 0.2% (v/v) DMSO and Triton X-100 were considered "no cell death" and "total cell death" control experiments, respectively, and incubated for 24 hours. Then, 20% (v/v) resazurin reagent (#G8080, Promega, USA) was added to the compound-treated cells and incubated for 2-3 hours. The relative amount of resorufin was quantified by measuring the fluorescence intensity at 590 nm. Finally, data were normalized to the control experiment and converted to “normalized % viability” as follows: (Experimental data-total cell death) / (No cell death-total cell death) × 100.

9. Statistical analysis

All experiments were performed at least three times independently. Results for multiple tests are presented as mean ± SD. Statistical analyzes were performed using Student's t-test or analysis of variance, as appropriate. Statistical significance was set at P<0.05. GraphPad Prism software was used to draw dose-response curves and calculate IC ₅₀ values.

<Experimental Example 1> Measurement of ANO1 induced current

In order to confirm that DES inhibits the ANO1 channel function even in single cells, a single cell patch clamp experiment was performed as in Experimental Protocol 2 above. ANO1 current was measured in HEK293 cells transiently expressing human ANO1, and the results are shown in FIGS. 1A and 1B .

1a and 1b, Ani9 (an ANO1 channel inhibitor) and diethylstilbestrol significantly inhibited ANO1-induced current by 77.7 ± 12.5% and 54.8 ± 16.7% at 1 μM and 10 μM, respectively. From this, it can be seen that diethylstilbestrol inhibits the activity of ANO1.

<Experimental Example 2> Measurement of ANO1 expression in various lung cancer cell lines

In order to measure the expression level of ANO1 in various lung cancer cell lines, as in experimental protocol 3, Western blotting for ANO1 protein was performed on FRT, FRT-hANO1, A549, H1993, HCC827, H1975, and PC9 cells. is shown in Figures 2a and 2b.

2a and 2b, unlike other lung cancer cell lines, it was confirmed that PC9 cells expressed ANO1 more than twice as strongly as FRT-hANO1 cells in which ANO1 was artificially overexpressed.

<Experimental Example 3> Analysis of ANO1 protein reduction effect in PC9 cells

When PC9 cells overexpressing ANO1 were treated with Eact, known as an ANO1 activator, Ani9, known as an ANO1 inhibitor, and diethylstilbestrol for 72 hours, the effect of reducing the ANO1 protein expression level was analyzed, and experimental protocol 3 was performed One result is shown in Figures 2c and 2d.

2c and 2d, in the case of Eact and Ani9 treatment, there is no change in the expression level of ANO1 protein, whereas in the case of treatment with 3 and 10 μM diethylstilbestrol, the expression level of ANO1 protein is ~ 48.5%, ~97% reduction is confirmed.

From this, it can be seen that diethylstilbestrol not only inhibits the activity of ANO1, but also reduces the expression of ANO1 protein.

<Experimental Example 4> Measurement of cell viability in H1975 and PC9 cells

As in the experimental protocol 4, the survival rate of each cell was measured when the H1975 cells, in which ANO1 was hardly expressed, and the PC9 cells, in which ANO1 was overexpressed, were treated with Eact, Ani9, and diethylstilbestrol, and the results is shown in Figures 3a and 3b.

3a and 3b, when Eact and Ani9 were treated, there was no change in cell viability in both H1975 cells and PC9 cells. On the other hand, when H1975 was treated with 3, 5, and 10 μM diethylstilbestrol, cell viability decreased by 16.1, 17.0, and 28.5%, respectively, and when PC9 was treated with 3, 5, and 10 μM diethylstilbestrol, It was confirmed that the cell viability decreased by 15.8, 51.8, and 85.2%, respectively.

From this, it can be seen that diethylstilbestrol exhibits anticancer activity selectively against lung cancer cells in which ANO1 is overexpressed. It is expected that it can be used as an effective anticancer agent.

<Experimental Example 5> Migration assay for PC9 cells

In order to confirm that diethylstilbestrol inhibits the migration of PC9 cells, as in Experimental Protocol 5, PC9 cells were treated with 3, 10 μM of diethylstilbestrol and 10 μM of Ani9, and cell migration inhibition was analyzed. , and the results are shown in Figs. 3c and 3d.

Referring to Figures 3c and 3d, it was confirmed that cell migration was reduced when PC9 cells were treated with diethylstilbestrol 3 or 10 µM, compared to when PC9 cells were treated with 10 µM of Ani9. Since diethylstilbestrol inhibits the migration of lung cancer cells, particularly ANO1-overexpressing lung cancer cells, diethylstilbestrol is expected to be useful not only as an anticancer agent for lung cancer, but also as a metastasis inhibitor.

<Experimental Example 6> Analysis of apoptosis effect of diethylstilbestrol on PC9 cells

We examined the mechanism through which diethylstilbestrol exhibits anticancer activity. In order to confirm the degree to which apoptosis is induced in PC9 cells, as in experimental protocols 6 and 7, PC9 cells were treated with Eact, Ani9, and diethylstilbestrol to induce caspase3 and cleaved PARP- known as apoptosis markers. The activation degree of 1 was measured, and the results are shown in FIGS. 4a and 4b.

4a and 4b, when PC9 cells were treated with Eact and Ani9, there was no change in the activation levels of caspase-3 and cleaved PARP-1. On the other hand, when diethylstilbestrol was treated at 3, 5, and 10 μM, the activation of caspase-3 increased 2, 4, and 6 times, respectively, and the activation of cleaved PARP-1 increased.

From this, it can be seen that diethylstilbestrol inhibits the expression of ANO1, thereby inducing apoptosis to exhibit anticancer activity.

<Experimental Example 7> Analysis of cell viability of diethylstilbestrol for hepatocytes

In order to confirm the development potential of diethylstilbestrol as a therapeutic agent for lung cancer, the toxicity of diethylstilbestrol to hepatocytes was confirmed as in Experimental Protocol 8 above. Hepatocytes (HepG2) were treated with Eact, Ani9 and diethylstilbestrol to measure cell viability, and the results are shown in FIG. 4c.

Referring to Figure 4c, compared to the case of Eact, Ani9 treatment of hepatocytes, it is confirmed that the cell viability of hepatocytes is not reduced up to 10 μM when diethylstilbestrol is treated.

Through this, diethylstilbestrol has anticancer activity against lung cancer, but is not toxic to normal hepatocytes even at high concentrations, so it is expected to be useful as an anticancer agent for lung cancer.

Claims (10)

A pharmaceutical composition for preventing or treating lung cancer containing diethylstilbestrol or a pharmaceutically acceptable salt thereof as an active ingredient. According to claim 1,
Characterized in that the pharmaceutical composition inhibits the expression of ANO1,
pharmaceutical composition. According to claim 1,
The lung cancer is characterized in that ANO1 is overexpressed,
pharmaceutical composition. According to claim 1,
Characterized in that the pharmaceutical composition induces apoptosis by inhibiting the expression of ANO1,
pharmaceutical composition. According to claim 1,
The lung cancer is non-small cell lung cancer,
pharmaceutical composition. A pharmaceutical composition for inhibiting lung cancer metastasis containing diethylstilbestrol or a pharmaceutically acceptable salt thereof as an active ingredient. According to claim 6,
Characterized in that the pharmaceutical composition inhibits the expression of ANO1,
pharmaceutical composition. According to claim 6,
The lung cancer is characterized in that ANO1 is overexpressed,
pharmaceutical composition. According to claim 6,
Characterized in that the pharmaceutical composition induces apoptosis by inhibiting the expression of ANO1,
pharmaceutical composition. According to claim 6,
The lung cancer is non-small cell lung cancer,
pharmaceutical composition.

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