KR102090554B1 - Radiosensitizer containing β-Apopicropodophyllin as an active ingredient - Google Patents

Abstract

The present invention relates to a novel use for the treatment of cancer of β-apopicropodophylline, which is a staphylococcal derivative, and more specifically, the present invention is administered to treat cancer in vivo to show an effect of enhancing cancer treatment. It relates to a new use of β- apopicropodophylline of formula 1, which can be.
[Formula 1]

Description

Radiosensitizer containing β-Apopicropodophyllin as an active ingredient

The present invention relates to a new use as a radiotherapy enhancer containing β-apopicropodophylline as an active ingredient, and more specifically, as an anti-cancer agent for treating cancer in vivo, as well as irradiating cancer cells with radiation The present invention relates to a radiation treatment enhancer comprising β-apopicropodophylline as an active ingredient, which can be used in combination with radiation treatment to enhance cancer treatment.

Humanity has continued to challenge cancer. Looking at the history of the development of anticancer drugs, so far, it has focused on the development of substances that kill cancer cells. Since the use of L-Azaserine isolated from Streptomyces in 1954 for the treatment of leukemia, mitomycin, bleomycin, anthramycin, and anthromycin ( daunomycin) has been developed and used for cancer treatment, and these anticancer agents are substances that kill cells, and have serious side effects as they are toxic to normal cells as well as cancer cells.

As such, conventional anticancer agents often kill even normal cells, and recently, studies on new mechanisms of anticancer agents that do not act on normal cells but can act on cancer cells are being conducted.

In addition, three types of typical anti-cancer treatment methods are surgical surgery, chemotherapy, and radiation therapy. Among them, about 50% of Korean solid cancer patients are currently receiving radiation therapy, and development and installation of new radiation therapy devices Through this, the number of cancer patients receiving radiation therapy is increasing every year. Also, in terms of the welfare of cancer patients, due to the side effects of cancer chemotherapy drugs and the burden of pain and loss of body function due to surgical procedures, the preference for radiation therapy, a relatively less painless and non-invasive treatment method, is increased. Therefore, the importance of radiotherapy in cancer treatment and its efficiency are required to be increased.

As described above, radiation treatment is currently known as an essential treatment method for various types of solid cancer, but problems such as side effects such as recurrence and metastasis due to radiation resistance acquisition, burns and fibrosis due to damage to normal tissues in the radiation treatment area are emerging. Came . Therefore, in order to solve these problems, it is necessary to increase the efficiency of radiation treatment using relatively low-dose radiation, and research on the development of an enhancer for achieving this has been ongoing.

[Patent Document 1] Korean Patent Publication No. 10-2005-0103259 [Patent Document 2] Korean Patent Application No. 10-2001-55544 [Patent Document 3] Korean Patent Application No. 10-2006-136639 [Patent Document 4] Korean Patent Application No. 10-2010-98596

The present invention has been devised to solve the problems of the prior art as described above, the object of the present invention is to provide an anti-cancer agent comprising β-apopicropodophylline as an active ingredient capable of effectively treating cancer at low concentrations. Is done.

In addition, an object of the present invention is to provide a radiotherapy enhancer comprising β-apopicropodophylline as an active ingredient that can reduce the side effects of radiation to normal cells while increasing the killing capacity of cancer cells at low concentrations. do.

In order to achieve the above object, the anti-cancer agent according to an embodiment of the present invention is characterized in that it contains β- apopiccropodophylline represented by the following formula (1) as an active ingredient.

[Formula 1]

The anticancer agent can be used for the treatment of lung cancer.

The concentration of β-apopicropodophylline as the anticancer agent may be 15 to 60 nM.

The anti-cancer agent may have an effect of inhibiting G2 / M cell cycle progression of cancer cells.

The anticancer agent may be an anticancer agent for oral administration or an anticancer agent for parenteral injection.

The parenteral injection may be selected from intravenous injection, intramuscular injection and subcutaneous injection.

When administering the anti-cancer agent for parenteral injection, β-apopicropodophylline may be administered in an amount of 1 to 5 mg / kg per time.

The anticancer agent is divided into a once-a-day administration schedule, and can be administered 2 to 3 times per week.

In one embodiment of the present invention, the radiotherapy enhancer according to the present invention is characterized in that it contains β-apopicropodophylline represented by the following Chemical Formula 1 as an active ingredient.

[Formula 1]

The radiation treatment may be lung cancer treatment by radiation.

The concentration of the β-apopicropodophylline is 10 to 20 nM, and radiation can be combined with radiation treatment by treating 1 to 5 Gy.

The radiation treatment enhancer may be divided into a dosing schedule once a day and administered twice to three times per week.

According to the present invention, not only has an anticancer effect by including a low concentration of β-apopicropodophylline as an active ingredient, but also has an effect of exhibiting an anticancer enhancing effect by being used in combination with radiation.

1 is a graph showing an increase in lung cancer cell death and IC50 value when treating β-apopicropodophylline with respect to human lung cancer cell A549.
Figure 2 is a graph showing the increase in lung cancer cell death and IC50 values in the treatment of β-apopicropodophylline against human lung cancer cells NCI-H1299.
Figure 3 is a graph showing the increase in lung cancer cell death and IC50 values in the treatment of β-apopicropodophylline with respect to human lung cancer cells NCI-H460.
FIG. 4 is a graph and photographs showing the results of lung cancer cell death in the treatment of human lung cancer cells A549, NCI-H1299 and NCI-H460 with staphylococcal toxin acetate and β-apopicropodophylline.
5 is a graph showing the results of cell death in the treatment of β-apopicropodophylline with respect to human lung cancer cells A549 and lung fibroblasts HFL1, WI-38 and IMR-90.
FIG. 6 is a graph showing the results of destruction of cell cycle regulation of cancer cells upon treatment of β-apopicropodophylline with respect to human lung cancer cells A549.
7 is a graph showing the result of destruction of cell cycle regulation of cancer cells when treatment with β-apopicropodophylline for human lung cancer cells NCI-H1299.
8 is a graph showing the destruction of the cell cycle regulation of cancer cells upon treatment of β-apopicropodophylline with respect to human lung cancer cells NCI-H460.
Figure 9 is a human lung cancer cells A549, NCI-H1299 and NCI-H460 for the treatment of β- apopiccropodophylline compared to the control (con) without any treatment compared to the results showing the increase in the damage of genetic material DNA It is a picture.
10 is a graph showing the result of increasing the cell death of cancer cells upon treatment of β-apopicropodophylline with respect to human lung cancer cells A549.
11 is a graph showing the result of increasing cell death of cancer cells when treating β-apopicropodophylline with respect to human lung cancer cells NCI-H1299.
12 is a graph showing the result of increasing the cell death of cancer cells upon treatment of β-apopicropodophylline with respect to human lung cancer cells NCI-H460.
13 is a photograph showing the result of the increase of major proteins of the ER stress signal that increases the cell death of cancer cells upon treatment of β-apopicropodophylline for human lung cancer cells A549.
FIG. 14 is a photograph showing the result of the increase of major proteins of the ER stress signal that increases cell death of cancer cells when treatment with β-apopicropodophylline for human lung cancer cells NCI-H1299.
FIG. 15 is a photograph showing the result of the increase of major proteins of the ER stress signal that increases the cell death of cancer cells upon treatment of β-apopicropodophylline with respect to human lung cancer cells NCI-H460.
16 is a graph showing the results of inhibition of tumor growth in mice by β-apopicropodophylline.
17 is a graph and photographs showing that the anti-cancer effect is increased upon treatment with β-apopicropodophylline and radiation for human lung cancer cells NCI-H1299.
18 is a graph and photographs showing that the anti-cancer effect is increased upon treatment with β-apopicropodophylline and radiation for human lung cancer cells NCI-H460.
19 is a graph showing that cell viability is reduced when human β-apochropodophylline and radiotherapy are used for human lung cancer cells NCI-H1299.
FIG. 20 is a graph showing that cell viability is reduced when the β-apopicropodophylline and radiotherapy are used for human lung cancer cells NCI-H460.
FIG. 21 is a photograph showing an increase in expression of proteins that are increased upon cell death in combination with β-apopicropodophylline and radiation for human lung cancer cells NCI-H1299.
FIG. 22 is a photograph showing an increase in expression of proteins that are increased upon cell death when β-apopicropodophylline and radiation are treated in combination with human lung cancer cells NCI-H460.
FIG. 23 is a graph and photographs showing the inhibition of tumor growth in mice by treatment with β-apopicropodophylline and radiotherapy for human lung cancer cells NCI-H460 and NCI-H1299.

Advantages and features of the present invention, and a method of achieving them will be clarified with reference to embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the specific embodiments disclosed below, but may be implemented in various different forms, and only the specific embodiments of the present invention allow the disclosure of the present invention to be complete, and are generally in the art to which the present invention pertains. It is provided to completely inform the person of knowledge of the scope of the invention, and the present invention is only defined by the scope of the claims.

Unless otherwise defined, all terms (including technical and scientific terms) used in the present specification may be used as meanings commonly understood by those skilled in the art to which the present invention pertains. In addition, terms defined in the commonly used dictionary are not ideally or excessively interpreted unless specifically defined.

Hereinafter, the anti-cancer agent and the radiation treatment enhancer according to the present invention will be described in detail.

The present inventors conducted experiments by administering β-apopicropodophylline as described above for various cancer cells. As a result, compared to the case where β-apopicropodophylline was not administered, cancer cell killing ability is enhanced, thereby improving therapeutic efficiency. It turns out that it can be maximized.

Β-apopicropodophylline5- (3,4,5-Trimethoxyphenyl) furo [3 ', 4': 6,7] naphtho [2,3-d] [1,3] usable as a therapeutic agent for cancer of the present invention ] dioxol-6 (9H) -one [β-Apopicropodophyllin, APP] is a compound having the structure represented by Chemical Formula 1, and is a derivative of podophyllotoxin [Journal of the American Chemical Society, 1988, Vol. 110, pp 7854-7858].

[Formula 1]

The β-apopicropodophylline can be prepared from grapephyllotoxin by culturing in ethanol buffer at high temperature as described in Buchardt, O. et al., J Pharmaceut Sci 75, 1076-1080, 1986. The total synthesis of picropodophylline and its apo derivatives is as described in Gensler, J.W., et al., J Am Chem Soc 82, 1714-1727, 1960.

The β-apopicropodophylline represented by Chemical Formula 1 has not been reported as a function of treating cancer.

In one embodiment of the present invention, the anti-cancer agent according to the present invention may include β-apopicropodophylline represented by Chemical Formula 1 as an active ingredient.

The anticancer agent can be used for the treatment of lung cancer.

The concentration of β-apopicropodophylline used as the anticancer agent is preferably 15 to 60 nM, more preferably 20 to 40 nM, and if it is less than 15 nM, the cytotoxicity is too weak and the anticancer effect is lost, which is undesirable. In addition, if it exceeds 60 nM, it may cause normal tissue damage, which is not preferable.

The anti-cancer agent may have an effect of inhibiting G2 / M cell cycle progression of cancer cells. The G2 group is a final step of preparing for division after DNA synthesis is completed, and the M group refers to a division stage.

The anticancer agent may be an anticancer agent for oral administration or an anticancer agent for parenteral injection.

When administering the anti-cancer drug for parenteral injection, β-apopicropodophylline may be administered in an amount of 1 to 5 mg / kg per time. It is undesirable, and if it exceeds 5 mg / kg, it causes toxicity to normal tissues, which is undesirable.

The anticancer agent is divided into a once-a-day administration schedule, and can be administered 2 to 3 times per week.

In another embodiment of the present invention, the radiotherapy enhancer according to the present invention may include β-apopicropodophylline represented by Chemical Formula 1 as an active ingredient.

The radiation treatment may be lung cancer treatment by radiation.

The concentration of β-apopicropodophylline used as the radiotherapy enhancer is preferably 10 to 20 nM, more preferably 10 to 15 nM, and if less than 10 nM, the efficacy of β-apopicropodophylline It is not preferable because it does not appear, and when it exceeds 20 nM, it is not preferable because the effect of killing cancer cells by the drug-only effect of β-apopicropodophylline is greater than the radiation effect.

The radiation can be used in combination with radiation treatment by treating 1 to 5 Gy, preferably 2 to 3 Gy. If it is less than 1 Gy, radiation effects do not appear, and if it exceeds 5 Gy, normal cell damage may occur. It is not desirable.

The radiation treatment enhancer may be divided into a dosing schedule once a day and administered twice to three times per week.

Hereinafter, the pharmacological action of β-apopicropodophylline according to the present invention will be described in more detail through experimental examples.

[Experimental Example 1]

In order to confirm that β-apopicropodophylline has an effect as an anticancer agent, the anticancer effect was tested as follows by treating β-apopicropodophylline (purchased from J & C Science, Korea) to lung cancer cells.

Human lung cancer cells A549, NCI-H1299 and NCl-H460 were cultured and used in RPMI 1640 medium containing 10% FBS and gentamicin.

1. Lung cancer cell killing effect test on treatment of β-apopicropodophylline against lung cancer cells

In order to treat lung cancer cells as one of the main targets of radiotherapy and having the highest mortality as a therapeutic target, MTT experiments were performed to obtain the results presented in FIGS. 1, 2 and 3. As a result of testing the cancer cell killing effect by APP using A549, NCI-H1299 and NCI-H460, the non-small cell lung cancer lines, the IC50 value of A549 was 16.86 nM, and the IC50 value of NCI-H1299 was 10.46 nM, In the case of NCI-H460, it showed an IC50 value of 17.09 nM, confirming that β-apopicropodophylline has the ability to kill lung cancer cells even at low concentrations. Therefore, it has been confirmed that β-apopicropodophylline, a grapephytotoxin derivative, exhibits anticancer activity by itself, and thus can be used as a therapeutic agent for cancer.

2. Analysis of cell viability after treatment with podophyllotoxin acetate and β-apopicropodophylline

In order to investigate how podophyllotoxin acetate (PA) and β-Apopicropodophyllin (APP) of the present invention affect cell death, cell viability assays were performed.

Human lung cancer cells A549, NCI-H1299, and NCI-H460 were each placed in approximately 30,000 6-well plates, and cultured for a day in a CO ₂ incubator at 37 ° C. Then, the treatment with podophyllotoxin acetate and β-apopicropodophylline of the present invention in A549, NCI-H1299 and NCI-H460 at a concentration of 40 nM and 60 nM, respectively, and incubated for 24 hours. Thereafter, 500 μl of a solution containing 1 μg / ml of propidium iodine was added, and cell death was analyzed using a FACSort flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). In addition, lung cancer cells A549, NCI-H1299, and NCI-H460 were each placed in a 100,000 mm 60 mm dish, and cultured for a day in a CO ₂ incubator at 37 ° C., followed by grapephyllotoxin acetate and β-apopicropodophylline. A549, NCI-H1299 and NCI-H460 were each treated at a concentration of 40 nM and incubated for 24 hours. And the cells of the group treated with only grapephyllotoxin acetate and the group treated only with β-apopicropodophylline of the present invention were collected and subjected to Western blot.

Analysis

Through the cell viability analysis experiment as described above, the cell killing effect by staphylococcal doplin and β- apopicropodophylline on human lung cancer cells is shown in FIG. 4.

As shown in Fig. 4 (A), when treating human lung cancer cells A549 with podophyllotoxin acetate and β-apopicropodophylline at 40 nM, the level of cell viability is higher than the control (Con) without any treatment. It was reduced to 98.3% and 82.3%, respectively, and it was observed that the β-apopicropodophylline-treated group decreased by 16% more than the grapephyllotoxin acetate-treated group. In addition, when treated with 60 nM of grapephytotoxin acetate and β-apopicropodophylline, compared to the control group without any treatment, the cell viability level decreased to 86.5% and 79.3%, respectively, and β-apopicophro It was confirmed that the phydophyllin-treated group decreased by 7.2% more than the phydophyllotoxin acetate-treated group.

As shown in FIG. 4 (B), when human lung cancer cells NCI-H1299 were treated with podophyllotoxin acetate and β-apopicropodophylline at 40 nM, the level of cell viability was higher than that of the control group without any treatment. It was reduced to 73.4% and 58.9%, respectively, and it was observed that the β-apopicropodophylline-treated group decreased 14.5% more than the grapephyllotoxin acetate-treated group. In addition, even when treated with 60 nM of podophyllotoxin acetate and β-apopicropodophylline, compared to the control group without any treatment, the cell viability levels decreased to 63.9% and 55.6%, respectively, and β-apopice It was confirmed that the ropodophylline-treated group decreased 8.3% more than the phydophyllotoxin acetate-treated group.

As shown in FIG. 4 (C), when treating human lung cancer cells NCI-H460 with podophyllotoxin acetate and β-apopicropodophylline at 40 nM, the level of cell viability than the control group without any treatment was It was reduced to 95.1% and 52.8%, respectively, and it was observed that the β-apopicropodophylline-treated group decreased by 42.3% more than the grapephyllotoxin acetate-treated group. In addition, when treated with 60 nM of podophyllotoxin acetate and β-apopicropodophylline, compared to the control group, cell viability levels decreased to 82.9% and 51.2%, respectively, and the β-apopicropodophylline treated group It was found that this was reduced by 31.7% more than the grapephytotoxin acetate treated group.

In addition, as shown in the lower part of FIG. 4, the group treated with β-apopicropodophylline at 40 nM for human lung cancer cells A549, NCI-H1299, and NCI-H460 through Western blot experiments was grapephytotoxin. It was observed that the protein expression of cleaved caspase-3 increased when cell death was increased compared to the control group treated with acetate at 40 nM. Therefore, through cell viability analysis experiments after treatment with staphylococcal doplin acetate and β-apopicropodophylline, it was verified that the death of cancer cells can be further increased when β-apopicropodophylline treatment, compared to staphylococcal doping treatment Did.

3. Biodetermination experiment of human lung cancer cells and lung fibroblasts by β-apopicropodophylline

Human cell lung cancer cells A549 and lung fibroblasts HFL1, WI-38 and IMR-90 were tested for cell survival biopsy as follows.

About 3,000 cells were placed in a 96-well plate and cultured for a day in a CO ₂ incubator at 37 ° C., followed by treatment with β-apopicropodophylline of the present invention at a concentration of 25 nM, followed by incubation for 24 and 48 hours, respectively. . Thereafter, 50 μl of a 2 mg / ml MTT (3- (4,5-dimethylthiazol-2-yle) -2,5-diphenyltetrazolium bromide) solution was treated, and then cultured for 2 hours. After removing the medium, 200 μl of DMSO was added, and absorbance was measured at 545 nm using a microplate reader.

Analysis

As shown in FIG. 5, when β-apopicropodophylline was treated, it was found that the cell viability of human lung cancer cells A549 was lower than that of lung fibroblasts HFL1, WI-38 and IMR-90. When β-apopicropodophylline was treated for 24 hours, human lung cancer cells A549 showed a survival rate of 35.2%, whereas lung fibroblasts HFL1, WI-38 and IMR-90 were 51.1% and 59.2, respectively. % And 79.1%.

In addition, when treated with β-apopicropodophylline for 48 hours, it was confirmed that the human lung cancer cell A549 showed a survival rate of 28.7%, and the pulmonary fibroblasts HFL1, WI-38 and IMR-90, respectively. It was confirmed that the survival rates of 54.5, 56.3 and 56.0% were shown. Therefore, through cell biodetermination experiments after β-apopicropodophylline treatment, it was verified that β-apopiccropodophylline shows a lower survival rate in human lung cancer cells than in lung fibroblasts.

4. Cell cycle assay

Human cycle cancer cells A549, NCI-H1299 and NCl-H460 were subjected to cell cycle analysis experiments as follows.

After about 300,000 of each of the cancer cells was placed in a 60 mm dish, and cultured for a day in a CO ₂ incubator at 37 ° C., β-apopicropodophylline of the present invention was 10 nM and 20 nM 2, respectively, in A549 and NCI-H460. After treatment with eggplant concentration, NCI-H1299 was treated with two different concentrations of 7.5 nM and 15 nM, followed by incubation for 8 hours. Then, it was fixed with ethanol and stained using RNase and propidium iodine, and the cell cycle was analyzed with a FACSort flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).

Analysis

Through the above cell cycle analysis experiment, the cell cycle arrest effect by β-apopicropodophylline on human lung cancer cells is shown in FIGS. 6, 7 and 8.

As shown in FIGS. 6, 7 and 8, when the β-apopicropodophylline was treated, G2 / M cell cycle arrest was observed. As shown in Fig. 6, the human lung cancer cell line A549 showed the highest G2 / M cell cycle effect when treated with 20 nM of β-apopicropodophylline, compared to the control group without any treatment. A 222% G2 / M cell cycle arrest effect was seen. And as shown in Figure 7, the human lung cancer cell line NCI-H1299 showed the highest G2 / M cell cycle effect when treated with 15 nM of β-apopicropodophylline, 165 than the control group without any treatment % G2 / M cell cycle arrest effect. In addition, as shown in FIG. 8, the human lung cancer cell line NCI-H460 showed the highest G2 / M cell cycle effect when treated with 20 nM of β-apopicropodophylline, 216.6% of G2 / M than the control group. It shows the cell cycle stopping effect.

5. DNA damage assay

Immunocytochemical staining experiments were performed to confirm the expression level of γ-H2AX protein related to damage to genetic material DNA.

Human lung cancer cells A549, NCI-H1299, and NCI-H460 are each placed in approximately 300,000 pieces in a 60 mm dish, and cultured for a day in a CO ₂ incubator at 37 ° C., followed by β-apopicropodophylline of the present invention, A549 and NCI-H460 After treating with a concentration of 20 nM, and treating with NCI-H1299 at a concentration of 15 nM, the cells were cultured for 48 hours each. Then, it was fixed with 1% paraformaldehyde and stained with anti-γ-H2AX antibody and DAPI. Pictures of dyed human lung cancer cells were obtained using a 710 confocal microscope (Carl-Zeiss, Germany), which is shown in FIG. 9.

Analysis

As shown in FIG. 9, after treatment with β-apopicropodophylline for human lung cancer cells A549, NCI-H1299, and NCI-H460, after 48 hours, γ-, unlike the control group without any treatment H2AX protein was observed in the form of dots in the nucleus. Therefore, it was confirmed through the immunocytochemical staining experiment that cell death due to DNA damage occurs.

6. Cell death assay

Whether cell death by β-apopicropodophylline occurs at the cell level as described above was confirmed through an apoptosis assay.

The cell death assay was performed by the FITC Annexin V Apoptosis Detection kit. I (BD Pharmingen, San Jose, CA, USA). Human lung cancer cells A549, NCI-H1299, and NCl-H460 are each placed in approximately 300,000 pieces in a 60 mm dish, and cultured for one day in a CO ₂ incubator at 37 ° C., followed by β-apopicropodophylline of the present invention, A549 and NCI- H460 was treated with a concentration of 20 nM, and NCI-H1299 was treated with a concentration of 15 nM, and then cultured for 48 hours. Thereafter, 500 μl of a solution containing 1 μg / ml of PI and Annexin V was added, and an apoptotic fraction was analyzed using a FACSort flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).

Analysis

Through the above cell death assay, the cell death effect by β-apopicropodophylline on human lung cancer cells is shown in FIGS. 10, 11 and 12.

As shown in Figure 10, when treated with human lung cancer cell A549, β-apopicropodophylline at 20 nM, the initial apoptosis level increased 28.6 times compared to the control group without any treatment, and the level of late apoptosis It was confirmed that the increase was 1.6 times.

As shown in Figure 11, when treated with β-apopicropodophylline at a concentration of 15 nM for human lung cancer cells NCI-H1299, the initial apoptosis level was increased by 20.3 times compared to the control group without any treatment, and late cells It was observed that the suicide level increased 4.3 times.

In addition, as shown in Figure 12, when treated with human lung cancer cells NCI-H460, β-apopicropodophylline at 20 nM, the initial apoptosis level was increased 5 times compared to the control group without any treatment, later It was confirmed that the apoptosis level increased 3.1 times.

7. Western blot experiment to verify the mechanism of signal transduction that induces apoptosis

The purpose of this study was to verify the cell death caused by TNFD at the cellular level as described above. Human lung cancer cells A549, NCI-H1299, and NCl-H460 are each placed in approximately 300,000 pieces in a 60 mm dish, and cultured for one day in a CO ₂ incubator at 37 ° C., followed by β-apopicropodophylline of the present invention, A549 and NCI- After treating at a concentration of 20 nM in H460 and treating at a concentration of 15 nM in NCI-H1299, the cells were cultured for 16 hours, 24 hours, and 48 hours, respectively. Then, cells were collected for each group to perform a Western blot experiment.

Analysis

Through the Western blot experiment for verifying the signal transduction mechanism as described above, the cell death effect by β-apopicropodophylline on human lung cancer cells is shown in FIGS. 13, 14, and 15.

As shown in FIG. 13, when treating β-apopicropodophylline at a concentration of 20 nM for human lung cancer cell A549, the major markers of ER stress, BiP, PDI, and phospho-PERK, are compared to the control group without any treatment. , It was confirmed that the protein expression levels of phospho-eIF2-α, ATF4, and CHOP increased over time.

As shown in FIG. 14, when human β-apochropodophylline is treated at a concentration of 15 nM for human lung cancer cells NCI-H1299, the major markers of ER stress, BiP, PDI, phospho-PERK, and phospho-eIF2, than the control group It was confirmed that the protein expression levels of -α, ATF4, and CHOP increased over time.

In addition, as shown in FIG. 15, when human β-apochropodophylline is treated with a concentration of 20 nM for human lung cancer cells NCI-H460, BiP, PDI, which are major markers of ER stress compared to the control group, compared to the control group, It was confirmed that the protein expression levels of phospho-PERK, phospho-eIF2-α, ATF4, and CHOP increased over time. Through the above-described signal transduction mechanism verification experiment, it was confirmed that continuous ER stress signaling by β-apopicropodophylline affects apoptosis.

8. Mouse tumor size change measurement and cancer cell death assay

The purpose of this study was to verify whether the anti-cancer effect of β-apopicropodophylline appears in mice as well as in cellular experiments.

Human lung cancer cells, NCI-H1299 and NCI-H460, were administered to subcutaneous tissue under the epidermis of mouse leg skin, respectively, about 10,000,000, and about 2,000,000, respectively, and examined for 2 weeks. Each tumor was approximately 100-200 mm ³ When this was done, the DMSO-treated group as a control group, the group treated with β-apopicropodophylline at 1 mg / kg, and the group treated with β-apopicropodophylline at 5 mg / kg were divided into three groups. DMSO, β-apopicropodophylline 1 mg / kg, β-apopicropodophylline 5 mg / kg were administered in 100 μl increments to the subcutaneous tissue under the epidermis of the mouse leg skin for 30 days. Changes in tumor size were examined. In addition, tumors were extracted by group and fixed with formaldehyde, and then the tumor tissues were produced by embedding them in paraffin blocks. The sliced tissue was then stained using the Apoptag TUNEL assay kit.

Analysis

Through the experiment for measuring the change in tumor size of mice inoculated with human lung cancer cells, the tumor growth delay by β-apopicropodophylline against human lung cancer cells NCI-H1299 and NCI-H460 is shown in FIG. 16.

As shown in FIG. 16, by evaluating the time required to reach the tumor volume of 800 mm ³ , it was shown that the β-apopicropodophylline treated group caused a growth delay compared to the DMSO treated group. As a result, the β-apopicropodophylline (1 mg / kg) group and β-apopiccropodophylline (5 mg / kg) group of mice inoculated with human lung cancer cells NCI-H1299 were compared to the control group inoculated with DMSO. Growth delays of 6.96 and 7.1 days, respectively. In addition, the β-apopicropodophylline (1 mg / kg) group and the β-apopiccropodophylline (5 mg / kg) group of mice inoculated with human lung cancer cell NCI-H460 compared to the control group for 6.11 days and Showed a growth delay of 10.64 days. In addition, the results of cell death analysis of xenograft tumor tissues using the Apoptag TUNEL assay kit are shown in the figure and quantified graphs. When the human lung cancer cell NCI-H1299 was transplanted xenograft, the β-apopicropodophylline (1 mg / kg) and β-apopiccropodophylline (5 mg / kg) groups were compared to the DMSO-inoculated control group. It was found that apoptotic cells increased by 9.3% and 24.0%, respectively. In addition, when the human lung cancer cell NCI-H1299 was transplanted xenograft, the β-apopicropodophylline (1 mg / kg) group and β-apopiccropodophylline (5 mg / kg) group had cell death compared to the control group. It was confirmed that the cells increased by 11.5% and 40.8%, respectively.

[Experimental Example 2]

To confirm whether β-apopicropodophylline has an effect as an anti-cancer enhancer, the anti-cancer enhancement effect was tested as follows by treating β-apopicropodophylline (purchased from J & C Science, Korea) to lung cancer cells.

Human lung cancer cells A549, NCI-H1299 and NCl-H460 were cultured and used in RPMI 1640 medium containing 10% FBS and gentamicin.

1.Clonogenicity assay after radiation treatment with β-apopicropodophylline

Human lung cancer cells, NCI-H1299 and NCl-H460, were subjected to clonogenic assay. About 100, about 200, about 400, about 600, and about 1000 cancer cells of NCI-H1299 and NCl-H460 were placed in a 60 mm dish, respectively, and cultured for a day in a CO ₂ incubator at 37 ° C. And, NCI-H1299 group treated with β-apopicropodophylline at a concentration of 10 nM and no treatment, NCI-H460 treated with a concentration of 15 nM and no treatment Split and incubate for 16 hours. Then, each group was exposed to radiation 1, 3, 5, and 7 Gy (gray, gray) of cesium-137. Then, after incubation for 10 to 14 days in a CO ₂ incubator at 37 ° C., colonies having a diameter of 200 μm or more were counted using a colony counter. Next, the cell-survival fraction was determined based on the number of colonies in the groups treated with and without treatment of β-apopicropodophylline.

Analysis

Through the above clonogenicity analysis experiment, the anti-cancer effect in combination treatment with β-apopicropodophylline and radiation is shown in FIGS. 17 and 18.

As shown in FIG. 17, DER calculated by comparing the cell survival fraction obtained based on the number of colonies of the group treated with radiation and β-apopicropodophylline and the control group treated only with radiation on human lung cancer cells NCI-H1299 (Dose-enhancement ratio) was 1.24.

As shown in FIG. 18, in the case of human lung cancer cell NCI-H460, DER calculated by comparing the cell survival fraction obtained based on the number of colonies between the control group treated with radiation only and the group treated with radiation and β-apopicropodophylline. Dose-enhancement ratio) was 1.44. Therefore, through the clonogenicity analysis experiment, it was confirmed that the anti-cancer effect was increased when the β-apopicropodophylline and radiotherapy were combined.

2. Cell viability assay after treatment with β-apopicropodophylline and radiation

In order to investigate how the combination treatment of β-apopicropodophylline and radiation at the cell level as described above affects cell death, a cell viability assay was performed.

About 30,000 human lung cancer cells NCI-H1299 and NCI-H460 were placed in 6-well plates, respectively, and cultured for one day in a CO ₂ incubator at 37 ° C, and then β-apopicropodophylline of the present invention was added to NCI-H1299 10. It was treated with a concentration of nM, and treated with a concentration of 15 nM in NCI-H460, and cultured for 16 hours each. After that, the radiation of cesium-137 was treated with 5 Gy for NCI-H1299 and 3 Gy for NCI-H460, and then cultured for 3 days. Then, 500 μl of a solution containing 1 μg / ml of PI was added, and cell death was analyzed using a FACSort flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). In addition, about 100,000 pieces of human lung cancer cells NCI-H1299 and NCI-H460 were placed in a 60 mm dish, and cultured for about a day in a CO ₂ incubator at 37 ° C., followed by β-apopicropodophylline of the present invention in 10 to NCI-H1299. It was treated with a concentration of nM, and treated with a concentration of 15 nM in NCI-H460, and each was cultured for 16 hours. Thereafter, the radiation of cesium-137 was treated with 5 Gy in NCI-H1299, and 3 Gy in NCI-H460, and then cultured for 3 days. In addition, cells of the control group, the group treated with only β-apopicropodophylline, the group treated with radiation only, and the group treated with β-apopicropodophylline and radiation were collected and subjected to Western blot.

Analysis

Through the cell viability analysis experiment as described above, the cell death effect by β-apopicropodophylline on human lung cancer cells is shown in FIGS. 19, 20, 21, and 22.

As shown in Figure 19, when treated with human lung cancer cells NCI-H1299, only β-apopicropodophylline at a concentration of 10 nM, the cell viability level was reduced to 71.5% than the control group without any treatment, When irradiated with only 5 Gy of radiation, it was observed that the cell viability level decreased to 73.8% compared to the control group without any treatment. In addition, when the combination of β-apopicropodophylline 10 nM and radiation 5 Gy, compared with the control group without any treatment, it was found that the cell viability level decreased to 42.3%.

In addition, as shown in FIG. 20, when treated with β-apopicropodophylline alone at a concentration of 15 nM for human lung cancer cells NCI-H460, the level of cell viability decreased to 78.9% compared to the control group without any treatment. In the case of irradiation with 3 Gy of radiation alone, it was observed that the cell viability level decreased to 89.5% compared to the control group without any treatment. In addition, when the combination of β-apopicropodophylline 15 nM and 3 Gy of radiation, compared with the control group without any treatment, it was found that the cell viability level decreased to 46.8%.

As shown in FIG. 21, β-apopicropodophylline and radiotherapy were performed on human lung cancer cells NCI-H1299 compared to the group treated with β-apopicropodophylline only at 10 nM and the group irradiated with only 5 Gy. It was observed that the protein expression of cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 increased when the cell death was compared to the control group.

As shown in FIG. 22, the human lung cancer cell NCI-H460 was treated with β-apopicropodophylline and radiation in combination with the group treated with β-apopicropodophylline at 15 nM and 3 Gy only with radiation. It was confirmed that the protein expression of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 increased when the cell death was compared to the control group.

Therefore, after treatment with β-apopicropodophylline and radiation, through cell viability assays, cancer cells were treated with β-apopicropodophylline and radiation in combination with β-apopicropodophylline and radiation alone. It was proved that the viability of the can be further reduced.

3. Measurement of tumor tumor size change and cancer cell killing assay in combination with β-apopicropodophylline and radiation

We tried to verify whether the anti-cancer effect of the combination treatment with β-apopicropodophylline and radiation appears in the mouse in vivo as well as on the cell.

As human lung cancer cells, approximately 10,000,000 and 5,000,000 NCI-H1299 and NCI-H460 were administered to the subcutaneous tissue under the epidermis of mouse leg skin, and after 2 weeks of examination, the size of each tumor was approximately 100-200 mm ³ . When done, DMSO as a control The treatment group, β-apopicropodophylline 5 mg / kg treatment group, and the radiation treatment group and β-apopicropodophylline and radiation combination treatment group was divided into three groups. DMSO and β-apopicropodophylline 5 mg / kg were administered to each subcutaneous tissue under the epidermis of mouse leg skin by 100 μl. In addition, the radiation-treated groups were treated with 5 Gy of radiation when human lung cancer cells NCI-H1299 were transplanted xenograft, and treated with 3 Gy of radiation when human lung cancer cell NCI-H460 was transplanted xenograft. Treatment groups with β-apopicropodophylline and radiation were treated with radiation 6 hours after administration of β-apopicropodophylline. Then, for about 30 days Changes in tumor size were examined. In addition, tumors were extracted for each group and fixed with formaldehyde, and then the tumor tissue was embedded in paraffin blocks. The sliced tissue was then stained using the Apoptag TUNEL assay kit.

Analysis

Through the experiment of measuring the change in tumor size of mice inoculated with human lung cancer cells as described above, human lung cancer cells were treated with β-apopicropodophylline alone, irradiated alone, and combined treatment with β-apopiccropodophylline and radiation. The tumor growth delay is shown in Figure 23. As shown in Fig. 23 (A), by evaluating the time required to reach the tumor volume of 800 mm ³ , the β-apopicropodophylline (5 mg / kg) alone treatment group and radiation (5 Gy) treatment alone It was shown that the group and the treatment groups combined with β-apopicropodophylline and radiation caused a growth delay compared to the DMSO treated group. As a result, β-apopicropodophylline (5 mg / kg) alone treatment group, radiation (5 Gy) alone treatment group, and β-apopiccropodophylline in combination with radiotherapy in mice inoculated with human lung cancer cell NCI-H1299 Treatment groups showed growth delays of 6.96 days, 7.1 days and 16.83 days, respectively, compared to DMSO treated controls.

As shown in FIG. 23 (B), β-apopicropodophylline (5 mg / kg) alone, radiation (5 Gy) alone, and β-aporeceptors in mice inoculated with human lung cancer cells NCI-H460 The groups treated with picropodophylline and radiation showed growth delays of 6.11, 10.64, and 22.38 days, respectively, compared to the DMSO-treated control group.

In addition, the results of cell death analysis of xenograft tumor tissues using the Apoptag TUNEL assay kit are shown in FIG. 23 (C, D) as a photograph and a quantified graph.

As shown in FIG. 23 (C), when human lung cancer cells NCI-H1299 were xenografted, β-apopicropodophylline (5 mg / kg) alone, radiation (5 Gy) alone, and β- It was found that the treatment groups with apopiccropodophylline and radiotherapy increased the cell death cells by 24.7%, 32.7% and 69.5%, respectively, compared to the control group.

 As shown in FIG. 23 (D), when human lung cancer cells NCI-H460 were heterologously transplanted, β-apopicropodophylline (5 mg / kg) alone, radiation (5 Gy) alone, and β- In the apopiccropodophylline and radiation combination treatment groups, it was confirmed that the cell death cells increased 38.8%, 50.3% and 112.3%, respectively, compared to the control group.

Claims (12)

delete delete delete delete delete delete delete delete A radiation treatment enhancer for treating lung cancer comprising β-apopicropodophylline represented by the following Chemical Formula 1 as an active ingredient.
[Formula 1]

delete The method of claim 9,
The concentration of β-apopicropodophylline is 10 to 20 nM, and the radiation is a radiotherapy enhancer for the treatment of lung cancer in combination with radiation treatment by treating 1 to 5 Gy. The method of claim 9,
The radiation treatment enhancer is a radiation treatment enhancer for lung cancer treatment, wherein the administration schedule is divided once a day and administered 2 to 3 times per week.

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